Antisense modulation of clusterin expression

ABSTRACT

Antisense compounds, compositions and methods are provided for modulating the expression of clusterin. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding clusterin. Methods of using these compounds for modulation of clusterin expression and for treatment of diseases associated with expression of clusterin are provided.

FIELD OF THE INVENTION

[0001] The present invention provides compositions and methods formodulating the expression of clusterin. In particular, this inventionrelates to compounds, particularly oligonucleotides, specificallyhybridizable with nucleic acids encoding clusterin. Such compounds havebeen shown to modulate the expression of clusterin.

BACKGROUND OF THE INVENTION

[0002] Clusterin is an amphipathic glycoprotein that was first isolatedfrom the male reproductive system (Bettuzzi et al., Biochem. J., 1989,257, 293-296; O'Bryan et al., J. Clin. Invest., 1990, 85, 1477-1486).Subsequently, it has been shown that clusterin is ubiquitouslydistributed among tissues, having a wide range of biologic properties.Investigators from several disciplines, therefore, have isolatedclusterin homologs under more than ten different names reviewed in(Bailey and Griswold, Mol. Cell. Endocrinol., 1999, 151, 17-23;Koch-Brandt and Morgans, Prog. Mol. Subcell. Biol., 1996, 16, 130-149;Meri and Jarva, Vox. Sang., 1998, 74, 291-302; Silkensen et al.,Biochem. Cell. Biol., 1994, 72, 483-488).

[0003] The clusterin protein consists of two non-identical subunits of34 kDa and 47 kDa, designated alpha and beta, respectively. Clusterinexpression is induced almost exclusively as a result of cellular injury,death, or pathology.

[0004] Among its many roles, clusterin is a component of the solubleSCb-5 complement complex which is assembled in the plasma uponactivation of the complement cascade (Choi et al., Mol. Immunol., 1989,26, 835-840; Kirszbaum et al., Embo J., 1989, 8, 711-718; Murphy et al.,Int. Immunol., 1989, 1, 551-554; Tschopp and French, Clin. Exp.Immunol., 1994, 97 Suppl 2, 11-14). Binding of clusterin has been shownto abolish the membranolytic potential of complement complexes and ithas therefore been termed complement lysis inhibitor (CLI) (Jenne andTschopp, Proc. Natl. Acad. Sci. U.S. A., 1989, 86, 7123-7127).

[0005] Further investigations of clusterin demonstrated that itcirculates in plasma as a high density lipoprotein (HDL) complex whichserves not only as an inhibitor of the lytic complement cascade, but asa regulator of lipid transport and local lipid redistribution (Jenne etal., J. Biol. Chem., 1991, 266, 11030-11036). In this capacity,clusterin isolated and characterized by de Silva et al. and was giventhe name Apolipoprotein J (ApoJ) (de Silva et al., Biochemistry, 1990,29, 5380-5389; de Silva et al., J. Biol. Chem., 1990, 265, 13240-13247;de Silva et al., J. Biol. Chem., 1990, 265, 14292-14297). In thesestudies, clusterin (ApoJ) was shown to play a role in cholesteroltransport in the liver and in the regulation of vascular smooth musclecell differentiation (de Silva et al., J. Biol. Chem., 1990, 265,13240-13247; Moulson and Millis, J. Cell. Physiol., 1999, 180, 355-364).A link between the modulation of HDL and complement activity is providedby studies by James et al. that characterize the association of a highdensity lipoprotein, NA1/NA2, with apolipoprotein A-I (ApoA-I). Thisnovel protein NA1/NA2, was subsequently shown to be clusterin (James etal., Arterioscler. Thromb., 1991, 11, 645-652.).

[0006] Clusterin has also been shown to participate in the cellularprocess of programmed cell death or apoptosis. Clusterin expressiondemarcates cells undergoing apoptosis (Buttyan et al., Mol. Cell. Biol.,1989, 9, 3473-3481) and in studies of the kidney, the onset ofhydronephrosis following unilateral obstruction is associated with theincreased expression of proteins encoded by the clusterin gene (Connoret al., Kidney Int., 1991, 39, 1098-1103). In both of these studies,clusterin is referred to by two other synonyms, sulfated glycoprotein-2gene (SGP-2) and testosterone-repressed prostate message-2 (TRPM-2)(Buttyan et al., Mol. Cell. Biol., 1989, 9, 3473-3481; Connor et al.,Kidney Int., 1991, 39, 1098-1103).

[0007] Sensibar et al. showed that cell death in the prostate, inducedby tumor necrosis factor alpha, could be prevented by overexpressingclusterin. In these studies, transfection of LNCaP cells with any offour 21-mer antisense phosphorothioate oligonucleotides targeting theclusterin coding region resulted in an increase of cell death (Sensibaret al., Cancer Res., 1995, 55, 2431-2437).

[0008] Miyake et al. further demonstrated the role of clusterin as ananti-apoptotic gene in the Shionogi tumor model, a model used for thestudy of castration-induced apoptosis (Miyake et al., Cancer Res., 2000,60, 170-176). In this model, androgen-dependent mammary carcinomaxenograft tumors in male mice undergo regression after castration butrecur as apoptosis-induced tumors after one month. Using aphosphorothioate 21-mer antisense oligonucleotide to the mouse clusteringene targeting the translation initiation site, Miyake et al. were ableto show that treatment with the clusterin antisense oligonucleotide ofmice with Shionogi tumors resulted in a more rapid onset of apoptosisand time to complete regresssion. There was also a significant delay ofemergence of androgen-independent recurrent tumors compared to controloligonucleotide treated controls.

[0009] Using the same oligonucleotide in an experiment designed to testthe efficacy of the oligonucleotide in combination with paclitaxel,Miyake et al. showed that the combination of antisense oligonucleotideand paclitaxel induced apoptosis in Shionogi tumors better than eitheragent alone. These studies suggest that the antisense oligonucleotidemay be useful in enhancing the effects of cytotoxic chemotherapy inhormone-refractory prostate cancer (Miyake et al., Cancer Res., 2000,60, 2547-2554). Ten antisense oligodeoxynucleotides targeted to humanTRPM-2 (clusterin) were designed by Miyake et al. (Clin. Cancer Res.,2000, 6, 1655-1663) to identify potent oligonucleotides thatspecifically inhibit TRPM-2 expression in human androgen-independentprostate cancer PC-2 cells. Seven of the ten oligonucleotides had littleor no effect on TRPM-2 mRNA expression. The other three oligonucleotideswere described by the authors as having moderate effects. The mostactive oligonucleotide was also tested for ability to enhance theresponse of PC-3 cells to Taxol or mitoxantrone.

[0010] Another antisense oligonucleotide, targeting the AUG initiationcodon of clusterin was used to investigate the role of clusterin inendothelial cell activation. In these studies, it was shown thatclusterin expression is upregulated upon laminar shear stress and thatreduction of clusterin levels via antisense treatment increasedendothelial cell activation (Urbich et al., Circulation, 2000, 101,352-355).

[0011] The level of clusterin is increased in the hippocampus andfrontal cortex of the brains of Alzheimer's disease patients. It iscurrently believed that clusterin, by binding to beta-amyloid, a proteinknown to aggregate in the brains of these patients, acts to link theprogression of this disease to the complement system (Choi-Miura andOda, Neurobiol. Aging, 1996, 17, 717-722).

[0012] Most recently, clusterin has been isolated as a KU70 bindingprotein. KU binding proteins (KUBs) are involved in DNA repair pathways.Clusterin (KUB1) was identified as an autoantigen in serum of patientswith scleroderma-polymyositis syndrome and shown to dimerize with KUP80to form an ATP dependent helicase and a regulatory component of a DNAdependent protein kinase (PRKDC) involved in double-strand break repairand V(D)J recombination (Yang et al., Nucleic Acids Res., 1999, 27,2165-2174).

[0013] Clusterin is overexpressed in many disease states includingneurodegenerative disorders, gliomas, retinitis pigmentosa andexpression is induced in acute and chronic models of renal injury anddisease, following ureter obstruction, ischemia/reperfusion, andatherosclerosis reviewed in (Silkensen et al., Biochem. Cell. Biol.,1994, 72, 483-488). The pharmacological modulation of clusterin activityand/or expression may therefore be an appropriate point of therapeuticintervention in pathological conditions.

[0014] The expression of clusterin, or variants thereof, has been usedas a means of differentiating normal versus abnormal cells in the studyof male infertility. A method of assessing acrosomal status of spermmorphology comprising contacting a sperm sample with an immunologicallyreactive molecule which binds to one form of clusterin and not anotheris disclosed in WO 95/16916.

[0015] Currently, there are no known therapeutic agents whicheffectively inhibit the synthesis of clusterin and to date,investigative strategies aimed at modulating clusterin function haveinvolved the use of antibodies, antisense oligonucleotides and chemicalinhibitors.

[0016] There remains, however, a long felt need for additional agentscapable of effectively inhibiting clusterin function.

[0017] Antisense technology is emerging as an effective means forreducing the expression of specific gene products and may thereforeprove to be uniquely useful in a number of therapeutic, diagnostic, andresearch applications for the modulation of clusterin expression.

[0018] The present invention provides compositions and methods formodulating clusterin expression, including modulation of the alphaand/or beta subunits.

SUMMARY OF THE INVENTION

[0019] The present invention is directed to compounds, particularlyantisense oligonucleotides, which are targeted to a nucleic acidencoding clusterin, and which modulate the expression of clusterin.Pharmaceutical and other compositions comprising the compounds of theinvention are also provided. Further provided are methods of modulatingthe expression of clusterin in cells or tissues comprising contactingsaid cells or tissues with one or more of the antisense compounds orcompositions of the invention. Further provided are methods of treatingan animal, particularly a human, suspected of having or being prone to adisease or condition associated with expression of clusterin byadministering a therapeutically or prophylactically effective amount ofone or more of the antisense compounds or compositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention employs oligomeric compounds, particularlyantisense oligonucleotides, for use in modulating the function ofnucleic acid molecules encoding clusterin, ultimately modulating theamount of clusterin produced. This is accomplished by providingantisense compounds which specifically hybridize with one or morenucleic acids encoding clusterin. As used herein, the terms “targetnucleic acid” and “nucleic acid encoding clusterin” encompass DNAencoding clusterin, RNA (including pre-mRNA and mRNA) transcribed fromsuch DNA, and also cDNA derived from such RNA. The specifichybridization of an oligomeric compound with its target nucleic acidinterferes with the normal function of the nucleic acid. This modulationof function of a target nucleic acid by compounds which specificallyhybridize to it is generally referred to as “antisense”. The functionsof DNA to be interfered with include replication and transcription. Thefunctions of RNA to be interfered with include all vital functions suchas, for example, translocation of the RNA to the site of proteintranslation, translation of protein from the RNA, splicing of the RNA toyield one or more mRNA species, and catalytic activity which may beengaged in or facilitated by the RNA. The overall effect of suchinterference with target nucleic acid function is modulation of theexpression of clusterin. In the context of the present invention,“modulation” means either an increase (stimulation) or a decrease(inhibition) in the expression of a gene. In the context of the presentinvention, inhibition is the preferred form of modulation of geneexpression and mRNA is a preferred target.

[0021] It is preferred to target specific nucleic acids for antisense.“Targeting” an antisense compound to a particular nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a nucleic acid molecule from aninfectious agent. In the present invention, the target is a nucleic acidmolecule encoding clusterin. The targeting process also includesdetermination of a site or sites within this gene for the antisenseinteraction to occur such that the desired effect, e.g., detection ormodulation of expression of the protein, will result. Within the contextof the present invention, a preferred intragenic site is the regionencompassing the translation initiation or termination codon of the openreading frame (ORF) of the gene. Since, as is known in the art, thetranslation initiation codon is typically 5′-AUG (in transcribed mRNAmolecules; 5′-ATG in the corresponding DNA molecule), the translationinitiation codon is also referred to as the “AUG codon,” the “startcodon” or the “AUG start codon”. A minority of genes have a translationinitiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, theterms “translation initiation codon” and “start codon” can encompassmany codon sequences, even though the initiator amino acid in eachinstance is typically methionine (in eukaryotes) or formylmethionine (inprokaryotes). It is also known in the art that eukaryotic andprokaryotic genes may have two or more alternative start codons, any oneof which may be preferentially utilized for translation initiation in aparticular cell type or tissue, or under a particular set of conditions.In the context of the invention, “start codon” and “translationinitiation codon” refer to the codon or codons that are used in vivo toinitiate translation of an mRNA molecule transcribed from a geneencoding clusterin, regardless of the sequence(s) of such codons.

[0022] It is also known in the art that a translation termination codon(or “stop codon”) of a gene may have one of three sequences, i.e.,5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA,5′-TAG and 5′-TGA, respectively). The terms “start codon region” and“translation initiation codon region” refer to a portion of such an mRNAor gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationinitiation codon. Similarly, the terms “stop codon region” and“translation termination codon region” refer to a portion of such anmRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationtermination codon.

[0023] The open reading frame (ORF) or “coding region,” which is knownin the art to refer to the region between the translation initiationcodon and the translation termination codon, is also a region which maybe targeted effectively. Other target regions include the 5′untranslated region (5′UTR), known in the art to refer to the portion ofan mRNA in the 5′ direction from the translation initiation codon, andthus including nucleotides between the 5′ cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene,and the 3′ untranslated region (3′UTR), known in the art to refer to theportion of an mRNA in the 3′ direction from the translation terminationcodon, and thus including nucleotides between the translationtermination codon and 3′ end of an mRNA or corresponding nucleotides onthe gene. The 5′ cap of an mRNA comprises an N7-methylated guanosineresidue joined to the 5′-most residue of the mRNA via a 5′-5′triphosphate linkage. The 5′ cap region of an mRNA is considered toinclude the 5′ cap structure itself as well as the first 50 nucleotidesadjacent to the cap. The 5′ cap region may also be a preferred targetregion.

[0024] Although some eukaryotic mRNA transcripts are directlytranslated, many contain one or more regions, known as “introns,” whichare excised from a transcript before it is translated. The remaining(and therefore translated) regions are known as “exons” and are splicedtogether to form a continuous mRNA sequence. mRNA splice sites, i.e.,intron-exon junctions, may also be preferred target regions, and areparticularly useful in situations where aberrant splicing is implicatedin disease, or where an overproduction of a particular mRNA spliceproduct is implicated in disease. Aberrant fusion junctions due torearrangements or deletions are also preferred targets. It has also beenfound that introns can also be effective, and therefore preferred,target regions for antisense compounds targeted, for example, to DNA orpre-mRNA.

[0025] Once one or more target sites have been identified,oligonucleotides are chosen which are sufficiently complementary to thetarget, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

[0026] In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases whichpair through the formation of hydrogen bonds. “Complementary,” as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. It is understood in the artthat the sequence of an antisense compound need not be 100%complementary to that of its target nucleic acid to be specificallyhybridizable. An antisense compound is specifically hybridizable whenbinding of the compound to the target DNA or RNA molecule interfereswith the normal function of the target DNA or RNA to cause a loss ofutility, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and in the case of in vitro assays, under conditions in whichthe assays are performed.

[0027] Antisense and other compounds of the invention which hybridize tothe target and inhibit expression of the target are identified throughexperimentation, and the sequences of these compounds are hereinbelowidentified as preferred embodiments of the invention. The target sitesto which these preferred sequences are complementary are hereinbelowreferred to as “active sites” and are therefore preferred sites fortargeting. Therefore another embodiment of the invention encompassescompounds which hybridize to these active sites.

[0028] Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes.Antisense compounds are also used, for example, to distinguish betweenfunctions of various members of a biological pathway. Antisensemodulation has, therefore, been harnessed for research use.

[0029] The specificity and sensitivity of antisense is also harnessed bythose of skill in the art for therapeutic uses. Antisenseoligonucleotides have been employed as therapeutic moieties in thetreatment of disease states in animals and man. Antisenseoligonucleotide drugs, including ribozymes, have been safely andeffectively administered to humans and numerous clinical trials arepresently underway. It is thus established that oligonucleotides can beuseful therapeutic modalities that can be configured to be useful intreatment regimes for treatment of cells, tissues and animals,especially humans.

[0030] In the context of this invention, the term “oligonucleotide”refers to an oligomer or polymer of ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA) or mimetics thereof. This term includesoligonucleotides composed of naturally-occurring nucleobases, sugars andcovalent internucleoside (backbone) linkages as well as oligonucleotideshaving non-naturally-occurring portions which function similarly. Suchmodified or substituted oligonucleotides are often preferred over nativeforms because of desirable properties such as, for example, enhancedcellular uptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

[0031] While antisense oligonucleotides are a preferred form ofantisense compound, the present invention comprehends other oligomericantisense compounds, including but not limited to oligonucleotidemimetics such as are described below. The antisense compounds inaccordance with this invention preferably comprise from about 8 to about50 nucleobases (i.e. from about 8 to about 50 linked nucleosides).Particularly preferred antisense compounds are antisenseoligonucleotides, even more preferably those comprising from about 12 toabout 30 nucleobases. Antisense compounds include ribozymes, externalguide sequence (EGS) oligonucleotides (oligozymes), and other shortcatalytic RNAs or catalytic oligonucleotides which hybridize to thetarget nucleic acid and modulate its expression.

[0032] As is known in the art, a nucleoside is a base-sugar combination.The base portion of the nucleoside is normally a heterocyclic base. Thetwo most common classes of such heterocyclic bases are the purines andthe pyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn the respective ends of this linear polymericstructure can be further joined to form a circular structure, however,open linear structures are generally preferred. Within theoligonucleotide structure, the phosphate groups are commonly referred toas forming the internucleoside backbone of the oligonucleotide. Thenormal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiesterlinkage.

[0033] Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

[0034] Preferred modified oligonucleotide backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates including3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acidforms are also included.

[0035] Representative United States patents that teach the preparationof the above phosphorus-containing linkages include, but are not limitedto, U.S. Pat. No. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050, certain of which are commonly owned with thisapplication, and each of which is herein incorporated by reference.

[0036] Preferred modified oligonucleotide backbones that do not includea phosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

[0037] Representative United States patents that teach the preparationof the above oligonucleosides include, but are not limited to, U.S. Pat.Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, certain of which are commonly ownedwith this application, and each of which is herein incorporated byreference.

[0038] In other preferred oligonucleotide mimetics, both the sugar andthe internucleoside linkage, i.e., the backbone, of the nucleotide unitsare replaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

[0039] Most preferred embodiments of the invention are oligonucleotideswith phosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂— ]of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

[0040] Modified oligonucleotides may also contain one or moresubstituted sugar moieties. Preferred oligonucleotides comprise one ofthe following at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, orN-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred areO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from1 to about 10. Other preferred oligonucleotides comprise one of thefollowing at the 2′ position: C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br,CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH2, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Apreferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim.Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples hereinbelow.

[0041] Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative United States patents that teach the preparationof such modified sugar structures include, but are not limited to, U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920, certain of which are commonly ownedwith the instant application, and each of which is herein incorporatedby reference in its entirety.

[0042] Oligonucleotides may also include nucleobase (often referred toin the art simply as “base”) modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat.No. 3,687,808, those disclosed in The Concise Encyclopedia Of PolymerScience And Engineering, pages 858-859, Kroschwitz, J. I., ed. JohnWiley & Sons, 1990, those disclosed by Englisch et al., AngewandteChemie, International Edition, 1991, 30, 613, and those disclosed bySanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain ofthese nucleobases are particularly useful for increasing the bindingaffinity of the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds., Antisense Research andApplications, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

[0043] Representative United States patents that teach the preparationof certain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; and 5,681,941, certain of which are commonly ownedwith the instant application, and each of which is herein incorporatedby reference, and U.S. Pat. No. 5,750,692, which is commonly owned withthe instant application and also herein incorporated by reference.

[0044] Another modification of the oligonucleotides of the inventioninvolves chemically linking to the oligonucleotide one or more moietiesor conjugates which enhance the activity, cellular distribution orcellular uptake of the oligonucleotide. Such moieties include but arenot limited to lipid moieties such as a cholesterol moiety (Letsinger etal., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid(Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), athioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad.Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let.,1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. AcidsRes., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol orundecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118;Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al.,Biochimie, 1993, 75, 49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937.

[0045] Representative United States patents that teach the preparationof such oligonucleotide conjugates include, but are not limited to, U.S.Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584;5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference.

[0046] It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds which are chimeric compounds. “Chimeric”antisense compounds or “chimeras,” in the context of this invention, areantisense compounds, particularly oligonucleotides, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of an oligonucleotide compound.These oligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the oligonucleotide may serve as a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way ofexample, RNase H is a cellular endonuclease which cleaves the RNA strandof an RNA:DNA duplex. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide inhibition of gene expression. Consequently, comparableresults can often be obtained with shorter oligonucleotides whenchimeric oligonucleotides are used, compared to phosphorothioatedeoxyoligonucleotides hybridizing to the same target region. Cleavage ofthe RNA target can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart.

[0047] Chimeric antisense compounds of the invention may be formed ascomposite structures of two or more oligonucleotides, modifiedoligonucleotides, oligonucleosides and/or oligonucleotide mimetics asdescribed above. Such compounds have also been referred to in the art ashybrids or gapmers. Representative United States patents that teach thepreparation of such hybrid structures include, but are not limited to,U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878;5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and5,700,922, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference inits entirety.

[0048] The antisense compounds used in accordance with this inventionmay be conveniently and routinely made through the well-known techniqueof solid phase synthesis. Equipment for such synthesis is sold byseveral vendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

[0049] The antisense compounds of the invention are synthesized in vitroand do not include antisense compositions of biological origin, orgenetic vector constructs designed to direct the in vivo synthesis ofantisense molecules.

[0050] The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes, receptortargeted molecules, oral, rectal, topical or other formulations, forassisting in uptake, distribution and/or absorption. RepresentativeUnited States patents that teach the preparation of such uptake,distribution and/or absorption assisting formulations include, but arenot limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

[0051] The antisense compounds of the invention encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to prodrugs and pharmaceutically acceptablesalts of the compounds of the invention, pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents.

[0052] The term “prodrug” indicates a therapeutic agent that is preparedin an inactive form that is converted to an active form (i.e., drug)within the body or cells thereof by the action of endogenous enzymes orother chemicals and/or conditions. In particular, prodrug versions ofthe oligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

[0053] The term “pharmaceutically acceptable salts” refers tophysiologically and pharmaceutically acceptable salts of the compoundsof the invention: i.e., salts that retain the desired biologicalactivity of the parent compound and do not impart undesiredtoxicological effects thereto.

[0054] Pharmaceutically acceptable base addition salts are formed withmetals or amines, such as alkali and alkaline earth metals or organicamines. Examples of metals used as cations are sodium, potassium,magnesium, calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred acid salts arethe hydrochlorides, acetates, salicylates, nitrates and phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfonic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds mayalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

[0055] For oligonucleotides, preferred examples of pharmaceuticallyacceptable salts include but are not limited to (a) salts formed withcations such as sodium, potassium, ammonium, magnesium, calcium,polyamines such as spermine and spermidine, etc.; (b) acid additionsalts formed with inorganic acids, for example hydrochloric acid,hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and thelike; (c) salts formed with organic acids such as, for example, aceticacid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaricacid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoicacid, tannic acid, palmitic acid, alginic acid, polyglutamic acid,naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

[0056] The antisense compounds of the present invention can be utilizedfor diagnostics, therapeutics, prophylaxis and as research reagents andkits. For therapeutics, an animal, preferably a human, suspected ofhaving a disease or disorder which can be treated by modulating theexpression of clusterin is treated by administering antisense compoundsin accordance with this invention. The compounds of the invention can beutilized in pharmaceutical compositions by adding an effective amount ofan antisense compound to a suitable pharmaceutically acceptable diluentor carrier. Use of the antisense compounds and methods of the inventionmay also be useful prophylactically, e.g., to prevent or delayinfection, inflammation or tumor formation, for example.

[0057] The antisense compounds of the invention are useful for researchand diagnostics, because these compounds hybridize to nucleic acidsencoding clusterin, enabling sandwich and other assays to easily beconstructed to exploit this fact. Hybridization of the antisenseoligonucleotides of the invention with a nucleic acid encoding clusterincan be detected by means known in the art. Such means may includeconjugation of an enzyme to the oligonucleotide, radiolabelling of theoligonucleotide or any other suitable detection means. Kits using suchdetection means for detecting the level of clusterin in a sample mayalso be prepared.

[0058] The present invention also includes pharmaceutical compositionsand formulations which include the antisense compounds of the invention.The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary, e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration. Oligonucleotides with at least one 2′-O-methoxyethylmodification are believed to be particularly useful for oraladministration.

[0059] Pharmaceutical compositions and formulations for topicaladministration may include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Coated condoms,gloves and the like may also be useful.

[0060] Compositions and formulations for oral administration includepowders or granules, suspensions or solutions in water or non-aqueousmedia, capsules, sachets or tablets. Thickeners, flavoring agents,diluents, emulsifiers, dispersing aids or binders may be desirable.

[0061] Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

[0062] Pharmaceutical compositions of the present invention include, butare not limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

[0063] The pharmaceutical formulations of the present invention, whichmay conveniently be presented in unit dosage form, may be preparedaccording to conventional techniques well known in the pharmaceuticalindustry. Such techniques include the step of bringing into associationthe active ingredients with the pharmaceutical carrier(s) orexcipient(s). In general the formulations are prepared by uniformly andintimately bringing into association the active ingredients with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

[0064] The compositions of the present invention may be formulated intoany of many possible dosage forms such as, but not limited to, tablets,capsules, liquid syrups, soft gels, suppositories, and enemas. Thecompositions of the present invention may also be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionsmay further contain substances which increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

[0065] In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

[0066] Emulsions

[0067] The compositions of the present invention may be prepared andformulated as emulsions. Emulsions are typically heterogenous systems ofone liquid dispersed in another in the form of droplets usuallyexceeding 0.1 μm in diameter. (Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 2, p. 335; Higuchi et al., in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p.301). Emulsions are often biphasic systems comprising of two immiscibleliquid phases intimately mixed and dispersed with each other. Ingeneral, emulsions may be either water-in-oil (w/o) or of theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions may contain additional componentsin addition to the dispersed phases and the active drug which may bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants may also be present in emulsions asneeded. Pharmaceutical emulsions may also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous provides an o/w/o emulsion.

[0068] Emulsions are characterized by little or no thermodynamicstability. Often, the dispersed or discontinuous phase of the emulsionis well dispersed into the external or continuous phase and maintainedin this form through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

[0069] Synthetic surfactants, also known as surface active agents, havefound wide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

[0070] Naturally occurring emulsifiers used in emulsion formulationsinclude lanolin, beeswax, phosphatides, lecithin and acacia. Absorptionbases possess hydrophilic properties such that they can soak up water toform w/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

[0071] A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0072] Hydrophilic colloids or hydrocolloids include naturally occurringgums and synthetic polymers such as polysaccharides (for example,acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, andtragacanth), cellulose derivatives (for example, carboxymethylcelluloseand carboxypropylcellulose), and synthetic polymers (for example,carbomers, cellulose ethers, and carboxyvinyl polymers). These disperseor swell in water to form colloidal solutions that stabilize emulsionsby forming strong interfacial films around the dispersed-phase dropletsand by increasing the viscosity of the external phase.

[0073] Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

[0074] The application of emulsion formulations via dermatological, oraland parenteral routes and methods for their manufacture have beenreviewed in the literature (Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 199). Emulsion formulations for oral deliveryhave been very widely used because of reasons of ease of formulation,efficacy from an absorption and bioavailability standpoint. (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

[0075] In one embodiment of the present invention, the compositions ofoligonucleotides and nucleic acids are formulated as microemulsions. Amicroemulsion may be defined as a system of water, oil and amphiphilewhich is a single optically isotropic and thermodynamically stableliquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 245). Typically microemulsions are systems that areprepared by first dispersing an oil in an aqueous surfactant solutionand then adding a sufficient amount of a fourth component, generally anintermediate chain-length alcohol to form a transparent system.Therefore, microemulsions have also been described as thermodynamicallystable, isotropically clear dispersions of two immiscible liquids thatare stabilized by interfacial films of surface-active molecules (Leungand Shah, in: Controlled Release of Drugs: Polymers and AggregateSystems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages185-215). Microemulsions commonly are prepared via a combination ofthree to five components that include oil, water, surfactant,cosurfactant and electrolyte. Whether the microemulsion is of thewater-in-oil (w/o) or an oil-in-water (o/w) type is dependent on theproperties of the oil and surfactant used and on the structure andgeometric packing of the polar heads and hydrocarbon tails of thesurfactant molecules (Schott, in Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0076] The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

[0077] Surfactants used in the preparation of microemulsions include,but are not limited to, ionic surfactants, non-ionic surfactants, Brij96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (S0750), decaglycerol decaoleate (DA0750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

[0078] Microemulsions are particularly of interest from the standpointof drug solubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or oligonucleotides. Microemulsions have also been effective inthe transdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucleotides and nucleic acidsfrom the gastrointestinal tract, as well as improve the local cellularuptake of oligonucleotides and nucleic acids within the gastrointestinaltract, vagina, buccal cavity and other areas of administration.

[0079] Microemulsions of the present invention may also containadditional components and additives such as sorbitan monostearate (Grill3), Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the oligonucleotides andnucleic acids of the present invention. Penetration enhancers used inthe microemulsions of the present invention may be classified asbelonging to one of five broad categories—surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

[0080] Liposomes

[0081] There are many organized surfactant structures besidesmicroemulsions that have been studied and used for the formulation ofdrugs. These include monolayers, micelles, bilayers and vesicles.Vesicles, such as liposomes, have attracted great interest because oftheir specificity and the duration of action they offer from thestandpoint of drug delivery. As used in the present invention, the term“liposome” means a vesicle composed of amphiphilic lipids arranged in aspherical bilayer or bilayers.

[0082] Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

[0083] In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

[0084] Further advantages of liposomes include; liposomes obtained fromnatural phospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

[0085] Liposomes are useful for the transfer and delivery of activeingredients to the site of action. Because the liposomal membrane isstructurally similar to biological membranes, when liposomes are appliedto a tissue, the liposomes start to merge with the cellular membranes.As the merging of the liposome and cell progresses, the liposomalcontents are emptied into the cell where the active agent may act.

[0086] Liposomal formulations have been the focus of extensiveinvestigation as the mode of delivery for many drugs. There is growingevidence that for topical administration, liposomes present severaladvantages over other formulations. Such advantages include reducedside-effects related to high systemic absorption of the administereddrug, increased accumulation of the administered drug at the desiredtarget, and the ability to administer a wide variety of drugs, bothhydrophilic and hydrophobic, into the skin.

[0087] Several reports have detailed the ability of liposomes to deliveragents including high-molecular weight DNA into the skin. Compoundsincluding analgesics, antibodies, hormones and high-molecular weightDNAs have been administered to the skin. The majority of applicationsresulted in the targeting of the upper epidermis.

[0088] Liposomes fall into two broad classes. Cationic liposomes arepositively charged liposomes which interact with the negatively chargedDNA molecules to form a stable complex. The positively chargedDNA/liposome complex binds to the negatively charged cell surface and isinternalized in an endosome. Due to the acidic pH within the endosome,the liposomes are ruptured, releasing their contents into the cellcytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147,980-985).

[0089] Liposomes which are pH-sensitive or negatively-charged, entrapDNA rather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

[0090] One major type of liposomal composition includes phospholipidsother than naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

[0091] Several studies have assessed the topical delivery of liposomaldrug formulations to the skin. Application of liposomes containinginterferon to guinea pig skin resulted in a reduction of skin herpessores while delivery of interferon via other means (e.g. as a solutionor as an emulsion) were ineffective (Weiner et al., Journal of DrugTargeting, 1992, 2, 405-410). Further, an additional study tested theefficacy of interferon administered as part of a liposomal formulationto the administration of interferon using an aqueous system, andconcluded that the liposomal formulation was superior to aqueousadministration (du Plessis et al., Antiviral Research, 1992, 18,259-265).

[0092] Non-ionic liposomal systems have also been examined to determinetheir utility in the delivery of drugs to the skin, in particularsystems comprising non-ionic surfactant and cholesterol. Non-ionicliposomal formulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).

[0093] Liposomes also include “sterically stabilized” liposomes, a termwhich, as used herein, refers to liposomes comprising one or morespecialized lipids that, when incorporated into liposomes, result inenhanced circulation lifetimes relative to liposomes lacking suchspecialized lipids. Examples of sterically stabilized liposomes arethose in which part of the vesicle-forming lipid portion of the liposome(A) comprises one or more glycolipids, such as monosialogangliosideG_(M1), or (B) is derivatized with one or more hydrophilic polymers,such as a polyethylene glycol (PEG) moiety. While not wishing to bebound by any particular theory, it is thought in the art that, at leastfor sterically stabilized liposomes containing gangliosides,sphingomyelin, or PEG-derivatized lipids, the enhanced circulationhalf-life of these sterically stabilized liposomes derives from areduced uptake into cells of the reticuloendothelial system (RES) (Allenet al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993,53, 3765). Various liposomes comprising one or more glycolipids areknown in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987,507, 64) reported the ability of monosialoganglioside G_(M1),galactocerebroside sulfate and phosphatidylinositol to improve bloodhalf-lives of liposomes. These findings were expounded upon by Gabizonet al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No.4,837,028 and WO 88/04924, both to Allen et al., disclose liposomescomprising (1) sphingomyelin and (2) the ganglioside G_(M1) or agalactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.)discloses liposomes comprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al.).

[0094] Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C₁₂15G, thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions-containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.).U.S. Pat. Nos. 5,540,935 (Miyazaki et al.) and 5,556,948 (Tagawa et al.)describe PEG-containing liposomes that can be further derivatized withfunctional moieties on their surfaces.

[0095] A limited number of liposomes comprising nucleic acids are knownin the art. WO 96/40062 to Thierry et al. discloses methods forencapsulating high molecular weight nucleic acids in liposomes. U.S.Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomesand asserts that the contents of such liposomes may include an antisenseRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methodsof encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Loveet al. discloses liposomes comprising antisense oligonucleotidestargeted to the raf gene.

[0096] Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g. they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

[0097] Surfactants find wide application in formulations such asemulsions (including microemulsions) and liposomes. The most common wayof classifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

[0098] If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

[0099] If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

[0100] If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

[0101] If the surfactant molecule has the ability to carry either apositive or negative charge, the surfactant is classified as amphoteric.Amphoteric surfactants include acrylic acid derivatives, substitutedalkylamides, N-alkylbetaines and phosphatides.

[0102] The use of surfactants in drug products, formulations and inemulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms,Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0103] Penetration Enhancers

[0104] In one embodiment, the present invention employs variouspenetration enhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides, to the skin of animals. Most drugs arepresent in solution in both ionized and nonionized forms. However,usually only lipid soluble or lipophilic drugs readily cross cellmembranes. It has been discovered that even non-lipophilic drugs maycross cell membranes if the membrane to be crossed is treated with apenetration enhancer. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs.

[0105] Penetration enhancers may be classified as belonging to one offive broad categories, i.e., surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Eachof the above mentioned classes of penetration enhancers are describedbelow in greater detail.

[0106] Surfactants: In connection with the present invention,surfactants (or “surface-active agents”) are chemical entities which,when dissolved in an aqueous solution, reduce the surface tension of thesolution or the interfacial tension between the aqueous solution andanother liquid, with the result that absorption of oligonucleotidesthrough the mucosa is enhanced. In addition to bile salts and fattyacids, these penetration enhancers include, for example, sodium laurylsulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetylether) (Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, p.92); and perfluorochemical emulsions, such as FC-43.Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

[0107] Fatty acids: Various fatty acids and their derivatives which actas penetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0108] Bile salts: The physiological role of bile includes thefacilitation of dispersion and absorption of lipids and fat-solublevitamins (Brunton, Chapter 38 in: Goodman & Gilman's The PharmacologicalBasis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, NewYork, 1996, pp. 934-935). Various natural bile salts, and theirsynthetic derivatives, act as penetration enhancers. Thus the term “bilesalts” includes any of the naturally occurring components of bile aswell as any of their synthetic derivatives. The bile salts of theinvention include, for example, cholic acid (or its pharmaceuticallyacceptable sodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee etal., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18thEd., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages782-783; Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992,263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[0109] Chelating Agents: Chelating agents, as used in connection withthe present invention, can be defined as compounds that remove metallicions from solution by forming complexes therewith, with the result thatabsorption of oligonucleotides through the mucosa is enhanced. Withregards to their use as penetration enhancers in the present invention,chelating agents have the added advantage of also serving as DNaseinhibitors, as most characterized DNA nucleases require a divalent metalion for catalysis and are thus inhibited by chelating agents (Jarrett,J. Chromatogr., 1993, 618, 315-339). Chelating agents of the inventioninclude but are not limited to disodium ethylenediaminetetraacetate(EDTA), citric acid, salicylates (e.g., sodium salicylate,5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen,laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

[0110] Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption ofoligonucleotides through the alimentary mucosa (Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This classof penetration enhancers include, for example, unsaturated cyclic ureas,1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92);and non-steroidal anti-inflammatory agents such as diclofenac sodium,indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol.,1987, 39, 621-626).

[0111] Agents that enhance uptake of oligonucleotides at the cellularlevel may also be added to the pharmaceutical and other compositions ofthe present invention. For example, cationic lipids, such as lipofectin(Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives,and polycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof oligonucleotides.

[0112] Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

[0113] Carriers

[0114] Certain compositions of the present invention also incorporatecarrier compounds in the formulation. As used herein, “carrier compound”or “carrier” can refer to a nucleic acid, or analog thereof, which isinert (i.e., does not possess biological activity per se) but isrecognized as a nucleic acid by in vivo processes that reduce thebioavailability of a nucleic acid having biological activity by, forexample, degrading the biologically active nucleic acid or promoting itsremoval from circulation. The coadministration of a nucleic acid and acarrier compound, typically with an excess of the latter substance, canresult in a substantial reduction of the amount of nucleic acidrecovered in the liver, kidney or other extracirculatory reservoirs,presumably due to competition between the carrier compound and thenucleic acid for a common receptor. For example, the recovery of apartially phosphorothioate oligonucleotide in hepatic tissue can bereduced when it is coadministered with polyinosinic acid, dextransulfate, polycytidic acid or4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al.,Antisense Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense &Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0115] Excipients

[0116] In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc.).

[0117] Pharmaceutically acceptable organic or inorganic excipientsuitable for non-parenteral administration which do not deleteriouslyreact with nucleic acids can also be used to formulate the compositionsof the present invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

[0118] Formulations for topical administration of nucleic acids mayinclude sterile and non-sterile aqueous solutions, non-aqueous solutionsin common solvents such as alcohols, or solutions of the nucleic acidsin liquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

[0119] Suitable pharmaceutically acceptable excipients include, but arenot limited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

[0120] Other Components

[0121] The compositions of the present invention may additionallycontain other adjunct components conventionally found in pharmaceuticalcompositions, at their art-established usage levels. Thus, for example,the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

[0122] Aqueous suspensions may contain substances which increase theviscosity of the suspension including, for example, sodiumcarboxymethylcellulose, sorbitol and/or dextran. The suspension may alsocontain stabilizers.

[0123] Certain embodiments of the invention provide pharmaceuticalcompositions containing (a) one or more antisense compounds and (b) oneor more other chemotherapeutic agents which function by a non-antisensemechanism. Examples of such chemotherapeutic agents include, but are notlimited to, anticancer drugs such as daunorubicin, dactinomycin,doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil,melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine(CA), 5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX),colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatinand diethylstilbestrol (DES). See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages 1206-1228). Anti-inflammatory drugs, including but notlimited to nonsteroidal anti-inflammatory drugs and corticosteroids, andantiviral drugs, including but not limited to ribivirin, vidarabine,acyclovir and ganciclovir, may also be combined in compositions of theinvention. See, generally, The Merck Manual of Diagnosis and Therapy,15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 2499-2506 and46-49, respectively). Other non-antisense chemotherapeutic agents arealso within the scope of this invention. Two or more combined compoundsmay be used together or sequentially.

[0124] In another related embodiment, compositions of the invention maycontain one or more antisense compounds, particularly oligonucleotides,targeted to a first nucleic acid and one or more additional antisensecompounds targeted to a second nucleic acid target. Numerous examples ofantisense compounds are known in the art. Two or more combined compoundsmay be used together or sequentially.

[0125] The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 ug to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 ug to 100 g per kgof body weight, once or more daily, to once every 20 years.

[0126] While the present invention has been described with specificityin accordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1

[0127] Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxyand 2′-alkoxy amidites

[0128] 2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropylphosphoramidites were purchased from commercial sources (e.g. Chemgenes,Needham Mass. or Glen Research, Inc. Sterling Va.). Other 2′-O-alkoxysubstituted nucleoside amidites are prepared as described in U.S. Pat.No. 5,506,351, herein incorporated by reference. For oligonucleotidessynthesized using 2′-alkoxy amidites, the standard cycle for unmodifiedoligonucleotides was utilized, except the wait step after pulse deliveryof tetrazole and base was increased to 360 seconds.

[0129] Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-C)nucleotides were synthesized according to published methods [Sanghvi,et. al., Nucleic Acids Research, 1993, 21, 3197-3203] using commerciallyavailable phosphoramidites (Glen Research, Sterling Va. or ChemGenes,Needham Mass.).

[0130] 2′-Fluoro Amidites

[0131] 2′-Fluorodeoxyadenosine Amidites

[0132] 2′-fluoro oligonucleotides were synthesized as describedpreviously [Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] andU.S. Pat. No. 5,670,633, herein incorporated by reference. Briefly, theprotected nucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine wassynthesized utilizing commercially available9-beta-D-arabinofuranosyladenine as starting material and by modifyingliterature procedures whereby the 2′-alpha-fluoro atom is introduced bya SN2-displacement of a 2′-beta-trityl group. ThusN6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively protected inmoderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate.Deprotection of the THP and N6-benzoyl groups was accomplished usingstandard methodologies and standard methods were used to obtain the5′-dimethoxytrityl-(DMT) and 5′-DMT-3′-phosphoramidite intermediates.

[0133] 2′-Fluorodeoxyguanosine

[0134] The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplishedusing tetraisopropyldisiloxanyl (TPDS) protected9-beta-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate diisobutyryl-arabinofuranosylguanosine. Deprotection ofthe TPDS group was followed by protection of the hydroxyl group with THPto give diisobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation was followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies were used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

[0135] 2′-Fluorouridine

[0136] Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by themodification of a literature procedure in which2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70%hydrogen fluoride-pyridine. Standard procedures were used to obtain the5′-DMT and 5′-DMT-3′phosphoramidites.

[0137] 2′-Fluorodeoxycytidine

[0138] 2′-deoxy-2′-fluorocytidine was synthesized via amination of2′-deoxy-2′-fluorouridine, followed by selective protection to giveN4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used toobtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

[0139] 2′-O-(2-Methoxyethyl) Modified Amidites

[0140] 2′-O-Methoxyethyl-substituted nucleoside amidites are prepared asfollows, or alternatively, as per the methods of Martin, P., HelveticaChimica Acta, 1995, 78, 486-504.

[0141]2,2′-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridine]

[0142] 5-Methyluridine (ribosylthymine, commercially available throughYamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate (90.0 g,0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300mL). The mixture was heated to reflux, with stirring, allowing theevolved carbon dioxide gas to be released in a controlled manner. After1 hour, the slightly darkened solution was concentrated under reducedpressure. The resulting syrup was poured into diethylether (2.5 L), withstirring. The product formed a gum. The ether was decanted and theresidue was dissolved in a minimum amount of methanol (ca. 400 mL). Thesolution was poured into fresh ether (2.5 L) to yield a stiff gum. Theether was decanted and the gum was dried in a vacuum oven (60° C. at 1mm Hg for 24 h) to give a solid that was crushed to a light tan powder(57 g, 85% crude yield). The NMR spectrum was consistent with thestructure, contaminated with phenol as its sodium salt (ca. 5%). Thematerial was used as is for further reactions (or it can be purifiedfurther by column chromatography using a gradient of methanol in ethylacetate (10-25%) to give a white solid, mp 222-4° C.).

[0143] 2′-O-Methoxyethyl-5-methyluridine

[0144] 2,2′-Anhydro-5-methyluridine (195 g, 0.81 M),tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol (1.2 L)were added to a 2 L stainless steel pressure vessel and placed in apre-heated oil bath at 160° C. After heating for 48 hours at 155-160°C., the vessel was opened and the solution evaporated to dryness andtriturated with MeOH (200 mL). The residue was suspended in hot acetone(1 L). The insoluble salts were filtered, washed with acetone (150 mL)and the filtrate evaporated. The residue (280 g) was dissolved in CH₃CN(600 mL) and evaporated. A silica gel column (3 kg) was packed inCH₂Cl₂/acetone/MeOH (20:5:3) containing 0.5% Et₃NH. The residue wasdissolved in CH₂Cl₂ (250 mL) and adsorbed onto silica (150 g) prior toloading onto the column. The product was eluted with the packing solventto give 160 g (63%) of product. Additional material was obtained byreworking impure fractions.

[0145] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

[0146] 2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) wasco-evaporated with pyridine (250 mL) and the dried residue dissolved inpyridine (1.3 L). A first aliquot of dimethoxytrityl chloride (94.3 g,0.278 M) was added and the mixture stirred at room temperature for onehour. A second aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the reaction stirred for an additional one hour. Methanol (170mL) was then added to stop the reaction. HPLC showed the presence ofapproximately 70% product. The solvent was evaporated and trituratedwith CH₃CN (200 mL). The residue was dissolved in CHCl₃ (1.5 L) andextracted with 2×500 mL of saturated NaHCO₃ and 2×500 mL of saturatedNaCl. The organic phase was dried over Na₂SO₄, filtered and evaporated.275 g of residue was obtained. The residue was purified on a 3.5 kgsilica gel column, packed and eluted with EtOAc/hexane/acetone (5:5:1)containing 0.5% Et₃NH. The pure fractions were evaporated to give 164 gof product. Approximately 20 g additional was obtained from the impurefractions to give a total yield of 183 g (57%).

[0147]3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

[0148] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g,0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL ofDMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M)were combined and stirred at room temperature for 24 hours. The reactionwas monitored by TLC by first quenching the TLC sample with the additionof MeOH. Upon completion of the reaction, as judged by TLC, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/hexane (4:1). Pure product fractions were evaporatedto yield 96 g (84%). An additional 1.5 g was recovered from laterfractions.

[0149]3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine

[0150] A first solution was prepared by dissolving3′-O-acetyl-2¹-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L),cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃was added dropwise, over a 30 minute period, to the stirred solutionmaintained at 0-10° C., and the resulting mixture stirred for anadditional 2 hours. The first solution was added dropwise, over a 45minute period, to the latter solution. The resulting reaction mixturewas stored overnight in a cold room. Salts were filtered from thereaction mixture and the solution was evaporated. The residue wasdissolved in EtOAc (1 L) and the insoluble solids were removed byfiltration. The filtrate was washed with 1×300 mL of NaHCO₃ and 2×300 mLof saturated NaCl, dried over sodium sulfate and evaporated. The residuewas triturated with EtOAc to give the title compound.

[0151] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

[0152] A solution of3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄OH (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH3 gas was added and the vesselheated to 100° C. for 2 hours (TLC showed complete conversion). Thevessel contents were evaporated to dryness and the residue was dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

[0153]N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

[0154] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyl-cytidine (85 g,0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g,0.165 M) was added with stirring. After stirring for 3 hours, TLC showedthe reaction to be approximately 95% complete. The solvent wasevaporated and the residue azeotroped with MeOH (200 mL). The residuewas dissolved in CHCl₃ (700 mL) and extracted with saturated NaHCO₃(2×300 mL) and saturated NaCl (2×300 mL), dried over MgSO₄ andevaporated to give a residue (96 g). The residue was chromatographed ona 1.5 kg silica column using EtOAc/hexane (1:1) containing 0.5% Et₃NH asthe eluting solvent. The pure product fractions were evaporated to give90 g (90%) of the title compound.

[0155]N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite

[0156]N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂Cl₂ (1 L) Tetrazole diisopropylamine (7.1g) and 2-cyanoethoxy-tetra(isopropyl)phosphite (40.5 mL, 0.123 M) wereadded with stirring, under a nitrogen atmosphere. The resulting mixturewas stirred for 20 hours at room temperature (TLC showed the reaction tobe 95% complete). The reaction mixture was extracted with saturatedNaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueous washes wereback-extracted with CH₂Cl₂ (300 mL), and the extracts were combined,dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc/hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g(87%) of the title compound.

[0157] 2′-O-(Aminooxyethyl) nucleoside amidites and2′-O-(dimethylaminooxyethyl) nucleoside amidites

[0158] 2′-(Dimethylaminooxyethoxy) nucleoside amidites

[0159] 2′-(Dimethylaminooxyethoxy) nucleoside amidites [also known inthe art as 2′-O-(dimethylaminooxyethyl) nucleoside amidites] areprepared as described in the following paragraphs. Adenosine, cytidineand guanosine nucleoside amidites are prepared similarly to thethymidine (5-methyluridine) except the exocyclic amines are protectedwith a benzoyl moiety in the case of adenosine and cytidine and withisobutyryl in the case of guanosine.

[0160] 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine

[0161] O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy,100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054mmol) were dissolved in dry pyridine (500 ml) at ambient temperatureunder an argon atmosphere and with mechanical stirring.tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol)was added in one portion. The reaction was stirred for 16 h at ambienttemperature. TLC (Rf 0.22, ethyl acetate) indicated a complete reaction.The solution was concentrated under reduced pressure to a thick oil.This was partitioned between dichloromethane (1 L) and saturated sodiumbicarbonate (2×1 L) and brine (1 L). The organic layer was dried oversodium sulfate and concentrated under reduced pressure to a thick oil.The oil was dissolved in a 1:1 mixture of ethyl acetate and ethyl ether(600 mL) and the solution was cooled to −10° C. The resultingcrystalline product was collected by filtration, washed with ethyl ether(3×200 mL) and dried (40° C., 1 mm Hg, 24 h) to 149 g (74.8%) of whitesolid. TLC and NMR were consistent with pure product.

[0162]5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

[0163] In a 2 L stainless steel, unstirred pressure reactor was addedborane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood andwith manual stirring, ethylene glycol (350 mL, excess) was addedcautiously at first until the evolution of hydrogen gas subsided.5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manualstirring. The reactor was sealed and heated in an oil bath until aninternal temperature of 160° C. was reached and then maintained for 16 h(pressure <100 psig). The reaction vessel was cooled to ambient andopened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T sideproduct, ethyl acetate) indicated about 70% conversion to the product.In order to avoid additional side product formation, the reaction wasstopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warmwater bath (40-100° C.) with the more extreme conditions used to removethe ethylene glycol. [Alternatively, once the low boiling solvent isgone, the remaining solution can be partitioned between ethyl acetateand water. The product will be in the organic phase.] The residue waspurified by column chromatography (2 kg silica gel, ethylacetate-hexanes gradient 1:1 to 4:1). The appropriate fractions werecombined, stripped and dried to product as a white crisp foam (84 g,50%), contaminated starting material (17.4 g) and pure reusable startingmaterial 20 g. The yield based on starting material less pure recoveredstarting material was 58%. TLC and NMR were consistent with 99% pureproduct.

[0164]2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

[0165]5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol)and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was then dried overP₂O₅ under high vacuum for two days at 40° C. The reaction mixture wasflushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) wasadded to get a clear solution. Diethyl-azodicarboxylate (6.98 mL, 44.36mmol) was added dropwise to the reaction mixture. The rate of additionis maintained such that resulting deep red coloration is just dischargedbefore adding the next drop. After the addition was complete, thereaction was stirred for 4 hrs. By that time TLC showed the completionof the reaction (ethylacetate:hexane, 60:40). The solvent was evaporatedin vacuum. Residue obtained was placed on a flash column and eluted withethyl acetate:hexane (60:40), to get2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine aswhite foam (21.819 g, 86%).

[0166]5′-O-tert-butyldiphenylsilyl-2-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

[0167]2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine(3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) andmethylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0°C. After 1 h the mixture was filtered, the filtrate was washed with icecold CH₂Cl₂ and the combined organic phase was washed with water, brineand dried over anhydrous Na₂SO₄. The solution was concentrated to get2′-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was addedand the resulting mixture was strirred for 1 h. Solvent was removedunder vacuum; residue chromatographed to get5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine as white foam (1.95 g, 78%).

[0168]5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine

[0169]5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine(1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridiniump-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride(0.39 g, 6.13 mmol) was added to this solution at 10° C. under inertatmosphere. The reaction mixture was stirred for 10 minutes at 10° C.After that the reaction vessel was removed from the ice bath and stirredat room temperature for 2 h, the reaction monitored by TLC (5% MeOH inCH₂Cl₂). Aqueous NaHCO₃ solution (5%, 10 mL) was added and extractedwith ethyl acetate (2×20 mL). Ethyl acetate phase was dried overanhydrous Na₂SO₄, evaporated to dryness. Residue was dissolved in asolution of 1M PPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL,3.37 mmol) was added and the reaction mixture was stirred at roomtemperature for 10 minutes. Reaction mixture cooled to 10° C. in an icebath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reactionmixture stirred at 10° C. for 10 minutes. After 10 minutes, the reactionmixture was removed from the ice bath and stirred at room temperaturefor 2 hrs. To the reaction mixture 5% NaHCO₃ (25 mL) solution was addedand extracted with ethyl acetate (2×25 mL). Ethyl acetate layer wasdried over anhydrous Na₂SO₄ and evaporated to dryness. The residueobtained was purified by flash column chromatography and eluted with 5%MeOH in CH₂Cl₂ to get5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridineas a white foam (14.6 g, 80%).

[0170] 2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0171] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolvedin dry THF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH).This mixture of triethylamine-2HF was then added to5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine(1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reactionwas monitored by TLC (5% MeOH in CH₂Cl₂). Solvent was removed undervacuum and the residue placed on a flash column and eluted with 10% MeOHin CH₂Cl₂ to get 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg,92.5%).

[0172] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0173] 2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol)was dried over P₂O₅ under high vacuum overnight at 40° C. It was thenco-evaporated with anhydrous pyridine (20 mL). The residue obtained wasdissolved in pyridine (11 mL) under argon atmosphere.4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4′-dimethoxytritylchloride (880 mg, 2.60 mmol) was added to the mixture and the reactionmixture was stirred at room temperature until all of the startingmaterial disappeared. Pyridine was removed under vacuum and the residuechromatographed and eluted with 10% MeOH in CH₂Cl₂ (containing a fewdrops of pyridine) to get5′-O-DMT--2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%).

[0174]5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0175] 5-O-DMT-2-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67mmol) was co-evaporated with toluene (20 mL). To the residueN,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and driedover P₂O₅ under high vacuum overnight at 40° C. Then the reactionmixture was dissolved in anhydrous acetonitrile (8.4 mL) and2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08mmol) was added. The reaction mixture was stirred at ambient temperaturefor 4 hrs under inert atmosphere. The progress of the reaction wasmonitored by TLC (hexane:ethyl acetate 1:1). The solvent was evaporated,then the residue was dissolved in ethyl acetate (70 mL) and washed with5% aqueous NaHCO₃ (40 mL). Ethyl acetate layer was dried over anhydrousNa₂SO₄ and concentrated. Residue obtained was chromatographed (ethylacetate as eluent) to get5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]as a foam (1.04 g, 74.9%).

[0176] 2′-(Aminooxyethoxy) nucleoside amidites

[0177] 2′-(Aminooxyethoxy) nucleoside amidites [also known in the art as2′-O-(aminooxyethyl) nucleoside amidites] are prepared as described inthe following paragraphs. Adenosine, cytidine and thymidine nucleosideamidites are prepared similarly.

[0178]N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0179] The 2′-O-aminooxyethyl guanosine analog may be obtained byselective 2′-O-alkylation of diaminopurine riboside. Multigramquantities of diaminopurine riboside may be purchased from Schering AG(Berlin) to provide 2′-O-(2-ethylacetyl) diaminopurine riboside alongwith a minor amount of the 3′-O-isomer. 2′-O-(2-ethylacetyl)diaminopurine riboside may be resolved and converted to2′-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase.(McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 A1 940203.)Standard protection procedures should afford2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine and2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosinewhich may be reduced to provide2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,41-dimethoxytrityl)guanosine.As before the hydroxyl group may be displaced by N-hydroxyphthalimidevia a Mitsunobu reaction, and the protected nucleoside mayphosphitylated as usual to yield2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,41-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

[0180] 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites

[0181] 2′-dimethylaminoethoxyethoxy nucleoside amidites (also known inthe art as 2′-O-dimethylaminoethoxyethyl, i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂,or 2′-DMAEOE nucleoside amidites) are prepared as follows. Othernucleoside amidites are prepared similarly.

[0182] 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine

[0183] 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) isslowly added to a solution of borane in tetra-hydrofuran (1 M, 10 mL, 10mmol) with stirring in a 100 mL bomb. Hydrogen gas evolves as the soliddissolves. O² -, 2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodiumbicarbonate (2.5 mg) are added and the bomb is sealed, placed in an oilbath and heated to 155° C. for 26 hours. The bomb is cooled to roomtemperature and opened. The crude solution is concentrated and theresidue partitioned between water (200 mL) and hexanes (200 mL). Theexcess phenol is extracted into the hexane layer. The aqueous layer isextracted with ethyl acetate (3×200 mL) and the combined organic layersare washed once with water, dried over anhydrous sodium sulfate andconcentrated. The residue is columned on silica gel usingmethanol/methylene chloride 1:20 (which has 2% triethylamine) as theeluent. As the column fractions are concentrated a colorless solid formswhich is collected to give the title compound as a white solid.

[0184] 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl uridine

[0185] To 0.5 g (1.3 mmol) of2′-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5-methyl uridine in anhydrouspyridine (8 mL), triethylamine (0.36 mL) and dimethoxytrityl chloride(DMT-Cl, 0.87 g, 2 eq.) are added and stirred for 1 hour. The reactionmixture is poured into water (200 mL) and extracted with CH₂Cl₂ (2×200mL). The combined CH₂Cl₂ layers are washed with saturated NaHCO₃solution, followed by saturated NaCl solution and dried over anhydroussodium sulfate. Evaporation of the solvent followed by silica gelchromatography using MeOH:CH₂Cl₂:Et₃N (20:1, v/v, with 1% triethylamine)gives the title compound.

[0186]5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite

[0187] Diisopropylaminotetrazolide (0.6 g) and2-cyanoethoxy-N,N-diisopropyl phosphoramidite (1.1 mL, 2 eq.) are addedto a solution of5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine(2.17 g, 3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere ofargon. The reaction mixture is stirred overnight and the solventevaporated. The resulting residue is purified by silica gel flash columnchromatography with ethyl acetate as the eluent to give the titlecompound.

Example 2

[0188] Oligonucleotide Synthesis

[0189] Unsubstituted and substituted phosphodiester (P═O)oligonucleotides are synthesized on an automated DNA synthesizer(Applied Biosystems model 380B) using standard phosphoramidite chemistrywith oxidation by iodine.

[0190] Phosphorothioates (P═S) are synthesized as for the phosphodiesteroligonucleotides except the standard oxidation bottle was replaced by0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrilefor the stepwise thiation of the phosphite linkages. The thiation waitstep was increased to 68 sec and was followed by the capping step. Aftercleavage from the CPG column and deblocking in concentrated ammoniumhydroxide at 55° C. (18 h), the oligonucleotides were purified byprecipitating twice with 2.5 volumes of ethanol from a 0.5 M NaClsolution. Phosphinate oligonucleotides are prepared as described in U.S.Pat. No. 5,508,270, herein incorporated by reference.

[0191] Alkyl phosphonate oligonucleotides are prepared as described inU.S. Pat. No. 4,469,863, herein incorporated by reference.

[0192] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are preparedas described in U.S. Pat. Nos. 5,610,289 or 5,625,050, hereinincorporated by reference.

[0193] Phosphoramidite oligonucleotides are prepared as described inU.S. Patent, 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporatedby reference.

[0194] Alkylphosphonothioate oligonucleotides are prepared as describedin published PCT applications PCT/US94/00902 and PCT/US93/06976(published as WO 94/17093 and WO 94/02499, respectively), hereinincorporated by reference.

[0195] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are preparedas described in U.S. Pat. No. 5,476,925, herein incorporated byreference.

[0196] Phosphotriester oligonucleotides are prepared as described inU.S. Pat. No. 5,023,243, herein incorporated by reference.

[0197] Borano phosphate oligonucleotides are prepared as described inU.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated byreference.

Example 3

[0198] Oligonucleoside Synthesis

[0199] Methylenemethylimino linked oligonucleosides, also identified asMMI linked oligonucleosides, methylenedi-methylhydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

[0200] Formacetal and thioformacetal linked oligonucleosides areprepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, hereinincorporated by reference.

[0201] Ethylene oxide linked oligonucleosides are prepared as describedin U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 4

[0202] PNA Synthesis

[0203] Peptide nucleic acids (PNAs) are prepared in accordance with anyof the various procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 5-23. They may also be prepared in accordance withU.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporatedby reference.

Example 5

[0204] Synthesis of Chimeric Oligonucleotides

[0205] Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[0206] [2′-O-Me]—[2′-deoxy]—[2′-O-Me] Chimeric PhosphorothioateOligonucleotides

[0207] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 380B, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.The standard synthesis cycle is modified by increasing the wait stepafter the delivery of tetrazole and base to 600 s repeated four timesfor RNA and twice for 2′-O-methyl. The fully protected oligonucleotideis cleaved from the support and the phosphate group is deprotected in3:1 ammonia/ethanol at room temperature overnight then lyophilized todryness. Treatment in methanolic ammonia for 24 hrs at room temperatureis then done to deprotect all bases and sample was again lyophilized todryness. The pellet is resuspended in 1M TBAF in THF for 24 hrs at roomtemperature to deprotect the 2′ positions. The reaction is then quenchedwith 1M TEAA and the sample is then reduced to ½ volume by rotovacbefore being desalted on a G25 size exclusion column. The oligorecovered is then analyzed spectrophotometrically for yield and forpurity by capillary electrophoresis and by mass spectrometry.

[0208] [2′-O-(2-Methoxyethyl)]—[2′-deoxy]—[2-O-(Methoxyethyl)] ChimericPhosphorothioate Oligonucleotides

[0209] [2′-O-(2-methoxyethyl)]—[2′-deoxy]—[-2′-O-(methoxyethyl)]chimeric phosphorothioate oligonucleotides were prepared as per theprocedure above for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

[0210] [2′-O-(2-Methoxyethyl)Phosphodiester]—[2′-deoxyPhosphorothioate]—[2′-O-(2-Methoxyethyl) Phosphodiester] ChimericOligonucleotides

[0211] [2′-O-(2-methoxyethyl phosphodiester]—[2′-deoxyphosphorothioate]—[2′-O-(methoxyethyl) phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidizationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

[0212] Other chimeric oligonucleotides, chimeric oligonucleosides andmixed chimeric oligonucleotides/oligonucleosides are synthesizedaccording to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6

[0213] Oligonucleotide Isolation

[0214] After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides or oligonucleosides are purified byprecipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol.Synthesized oligonucleotides were analyzed by polyacrylamide gelelectrophoresis on denaturing gels and judged to be at least 85% fulllength material. The relative amounts of phosphorothioate andphosphodiester linkages obtained in synthesis were periodically checkedby ³¹P nuclear magnetic resonance spectroscopy, and for some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7

[0215] Oligonucleotide Synthesis—96 Well Plate Format

[0216] Oligonucleotides were synthesized via solid phase P(III)phosphoramidite chemistry on an automated synthesizer capable ofassembling 96 sequences simultaneously in a standard 96 well format.Phosphodiester internucleotide linkages were afforded by oxidation withaqueous iodine. Phosphorothioate internucleotide linkages were generatedby sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyldiisopropyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per known literature or patented methods. They are utilized as baseprotected beta-cyanoethyldiisopropyl phosphoramidites.

[0217] Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8

[0218] Oligonucleotide Analysis—96 Well Plate Format

[0219] The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96 well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman p/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 9

[0220] Cell culture and oligonucleotide treatment

[0221] The effect of antisense compounds on target nucleic acidexpression can be tested in any of a variety of cell types provided thatthe target nucleic acid is present at measurable levels. This can beroutinely determined using, for example, PCR or Northern blot analysis.The following 4 cell types are provided for illustrative purposes, butother cell types can be routinely used, provided that the target isexpressed in the cell type chosen. This can be readily determined bymethods routine in the art, for example Northern blot analysis,Ribonuclease protection assays, or RT-PCR.

[0222] T-24 Cells:

[0223] The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10%fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.),penicillin 100 units per mL, and streptomycin 100 micrograms per mL(Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinelypassaged by trypsinization and dilution when they reached 90%confluence. Cells were seeded into 96-well plates (Falcon-Primaria#3872) at a density of 7000 cells/well for use in RT-PCR analysis.

[0224] For Northern blotting or other analysis, cells may be seeded onto100 mm or other standard tissue culture plates and treated similarly,using appropriate volumes of medium and oligonucleotide.

[0225] A549 Cells:

[0226] The human lung carcinoma cell line A549 was obtained from theAmerican Type Culture Collection (ATCC) (Manassas, VA). A549 cells wereroutinely cultured in DMEM basal media (Gibco/Life Technologies,Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/LifeTechnologies, Gaithersburg, Md.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Gibco/Life Technologies,Gaithersburg, Md.). Cells were routinely passaged by trypsinization anddilution when they reached 90% confluence.

[0227] NHDF Cells:

[0228] Human neonatal dermal fibroblast (NHDF) were obtained from theClonetics Corporation (Walkersville Md.). NHDFs were routinelymaintained in Fibroblast Growth Medium (Clonetics Corporation,Walkersville Md.) supplemented as recommended by the supplier. Cellswere maintained for up to 10 passages as recommended by the supplier.

[0229] HEK Cells:

[0230] Human embryonic keratinocytes (HEK) were obtained from theClonetics Corporation (Walkersville Md.). HEKs were routinely maintainedin Keratinocyte Growth Medium (Clonetics Corporation, Walkersville Md.)formulated as recommended by the supplier. Cells were routinelymaintained for up to 10 passages as recommended by the supplier.

[0231] Treatment With Antisense Compounds:

[0232] When cells reached 80% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 200 μL OPTI-MEM™-1 reduced-serum medium (Gibco BRL) and thentreated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™(Gibco BRL) and the desired concentration of oligonucleotide. After 4-7hours of treatment, the medium was replaced with fresh medium. Cellswere harvested 16-24 hours after oligonucleotide treatment.

[0233] The concentration of oligonucleotide used varies from cell lineto cell line. To determine the optimal oligonucleotide concentration fora particular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is ISIS 13920, TCCGTCATCGCTCCTCAGGG,SEQ ID NO: 1, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown inbold) with a phosphorothioate backbone which is targeted to human H-ras.For mouse or rat cells the positive control oligonucleotide is ISIS15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 2, a 2′-O-methoxyethyl gapmer(2′-O-methoxyethyls shown in bold) with a phosphorothioate backbonewhich is targeted to both mouse and rat c-raf. The concentration ofpositive control oligonucleotide that results in 80% inhibition ofc-Ha-ras (for ISIS 13920) or c-raf (for ISIS 15770) mRNA is thenutilized as the screening concentration for new oligonucleotides insubsequent experiments for that cell line. If 80% inhibition is notachieved, the lowest concentration of positive control oligonucleotidethat results in 60% inhibition of H-ras or c-raf mRNA is then utilizedas the oligonucleotide screening concentration in subsequent experimentsfor that cell line. If 60% inhibition is not achieved, that particularcell line is deemed as unsuitable for oligonucleotide transfectionexperiments.

Example 10

[0234] Analysis of Oligonucleotide Inhibition of Clusterin Expression

[0235] Antisense modulation of clusterin expression can be assayed in avariety of ways known in the art. For example, clusterin mRNA levels canbe quantitated by, e.g., Northern blot analysis, competitive polymerasechain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitativePCR is presently preferred. RNA analysis can be performed on totalcellular RNA or poly(A)+ mRNA. Methods of RNA isolation are taught in,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons,Inc., 1993. Northern blot analysis is routine in the art and is taughtin, for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996.Real-time quantitative (PCR) can be conveniently accomplished using thecommercially available ABI PRISM™ 7700 Sequence Detection System,available from PE-Applied Biosystems, Foster City, Calif. and usedaccording to manufacturer's instructions. Prior to quantitative PCRanalysis, primer-probe sets specific to the target gene being measuredare evaluated for their ability to be “multiplexed” with a GAPDHamplification reaction. In multiplexing, both the target gene and theinternal standard gene GAPDH are amplified concurrently in a singlesample. In this analysis, mRNA isolated from untreated cells is seriallydiluted. Each dilution is amplified in the presence of primer-probe setsspecific for GAPDH only, target gene only (“single-plexing”), or both(multiplexing). Following PCR amplification, standard curves of GAPDHand target mRNA signal as a function of dilution are generated from boththe single-plexed and multiplexed samples. If both the slope andcorrelation coefficient of the GAPDH and target signals generated fromthe multiplexed samples fall within 10% of their corresponding valuesgenerated from the single-plexed samples, the primer-probe set specificfor that target is deemed as multiplexable. Other methods of PCR arealso known in the art.

[0236] Protein levels of clusterin can be quantitated in a variety ofways well known in the art, such as immunoprecipitation, Western blotanalysis (immunoblotting), ELISA or fluorescence-activated cell sorting(FACS). Antibodies directed to clusterin can be identified and obtainedfrom a variety of sources, such as the MSRS catalog of antibodies (AerieCorporation, Birmingham, Mich.), or can be prepared via conventionalantibody generation methods. Methods for preparation of polyclonalantisera are taught in, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, JohnWiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is taughtin, for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997.

[0237] Immunoprecipitation methods are standard in the art and can befound at, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons,Inc., 1998. Western blot (immunoblot) analysis is standard in the artand can be found at, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley& Sons, Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) arestandard in the art and can be found at, for example, Ausubel, F. M. etal., Current Protocols in Molecular Biology, Volume 2, pp.11.2.1-11.2.22, John Wiley & Sons, Inc., 1991.

Example 11

[0238] Poly(A)+ mRNA Isolation

[0239] Poly(A)+ mRNA was isolated according to Miura et al., Clin.Chem., 1996, 42, 1758-1764. Other methods for poly(A)+ mRNA isolationare taught in, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc.,1993. Briefly, for cells grown on 96-well plates, growth medium wasremoved from the cells and each well was washed with 200 μL cold PBS. 60μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5%NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, theplate was gently agitated and then incubated at room temperature forfive minutes. 55 μL of lysate was transferred to Oligo d(T) coated96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60minutes at room temperature, washed 3 times with 200 μL of wash buffer(10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash,the plate was blotted on paper towels to remove excess wash buffer andthen air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH7.6), preheated to 70° C. was added to each well, the plate wasincubated on a 90° C. hot plate for 5 minutes, and the eluate was thentransferred to a fresh 96-well plate.

[0240] Cells grown on 100 mm or other standard plates may be treatedsimilarly, using appropriate volumes of all solutions.

Example 12

[0241] Total RNA Isolation

[0242] Total mRNA was isolated using an RNEASY 96™ kit and bufferspurchased from Qiagen Inc. (Valencia Calif.) following themanufacturer's recommended procedures. Briefly, for cells grown on96-well plates, growth medium was removed from the cells and each wellwas washed with 200 μL cold PBS. 100 μL Buffer RLT was added to eachwell and the plate vigorously agitated for 20 seconds. 100 μL of 70%ethanol was then added to each well and the contents mixed by pipettingthree times up and down. The samples were then transferred to the RNEASY96™ well plate attached to a QIAVAC™ manifold fitted with a wastecollection tray and attached to a vacuum source. Vacuum was applied for15 seconds. 1 mL of Buffer RW1 was added to each well of the RNEASY 96™plate and the vacuum again applied for 15 seconds. 1 mL of Buffer RPEwas then added to each well of the RNEASY 96™ plate and the vacuumapplied for a period of 15 seconds. The Buffer RPE wash was thenrepeated and the vacuum was applied for an additional 10 minutes. Theplate was then removed from the QIAVAC™ manifold and blotted dry onpaper towels. The plate was then re-attached to the QIAVAC™ manifoldfitted with a collection tube rack containing 1.2 mL collection tubes.RNA was then eluted by pipetting 60 μL water into each well, incubating1 minute, and then applying the vacuum for 30 seconds. The elution stepwas repeated with an additional 60 μL water.

[0243] The repetitive pipetting and elution steps may be automated usinga QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 13

[0244] Real-Time Quantitative PCR Analysis of Clusterin mRNA Levels

[0245] Quantitation of clusterin mRNA levels was determined by real-timequantitative PCR using the ABI PRISM™ 7700 Sequence Detection System(PE-Applied Biosystems, Foster City, Calif.) according to manufacturer'sinstructions. This is a closed-tube, non-gel-based, fluorescencedetection system which allows high-throughput quantitation of polymerasechain reaction (PCR) products in real-time. As opposed to standard PCR,in which amplification products are quantitated after the PCR iscompleted, products in real-time quantitative PCR are quantitated asthey accumulate. This is accomplished by including in the PCR reactionan oligonucleotide probe that anneals specifically between the forwardand reverse PCR primers, and contains two fluorescent dyes. A reporterdye (e.g., JOE, FAM, or VIC, obtained from either Operon TechnologiesInc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) isattached to the 5′ end of the probe and a quencher dye (e.g., TAMRA,obtained from either Operon Technologies Inc., Alameda, Calif. orPE-Applied Biosystems, Foster City, Calif.) is attached to the 3′ end ofthe probe. When the probe and dyes are intact, reporter dye emission isquenched by the proximity of the 3′ quencher dye. During amplification,annealing of the probe to the target sequence creates a substrate thatcan be cleaved by the 5′-exonuclease activity of Taq polymerase. Duringthe extension phase of the PCR amplification cycle, cleavage of theprobe by Taq polymerase releases the reporter dye from the remainder ofthe probe (and hence from the quencher moiety) and a sequence-specificfluorescent signal is generated. With each cycle, additional reporterdye molecules are cleaved from their respective probes, and thefluorescence intensity is monitored at regular intervals by laser opticsbuilt into the ABI PRISM™ 7700 Sequence Detection System. In each assay,a series of parallel reactions containing serial dilutions of mRNA fromuntreated control samples generates a standard curve that is used toquantitate the percent inhibition after antisense oligonucleotidetreatment of test samples.

[0246] PCR reagents were obtained from PE-Applied Biosystems, FosterCity, Calif. RT-PCR reactions were carried out by adding 25 μL PCRcocktail (1×TAQMAN™ buffer A, 5.5 mM MgCl₂, 300 μM each of dATP, dCTPand dGTP, 600 μM of dUTP, 100 nM each of forward primer, reverse primer,and probe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD™, and 12.5Units MULV reverse transcriptase) to 96 well plates containing 25 μLpoly(A) mRNA solution. The RT reaction was carried out by incubation for30 minutes at 48° C. Following a 10 minute incubation at 95° C. toactivate the AMPLITAQ GOLD™, 40 cycles of a two-step PCR protocol werecarried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for1.5 minutes (annealing/extension).

[0247] Probes and primers to human clusterin were designed to hybridizeto a human clusterin sequence, using published sequence information(GenBank accession number M64722, incorporated herein as SEQ ID NO:3).For human clusterin the PCR primers were:

[0248] forward primer: TCCGTACGAGCCCCTGAA (SEQ ID NO: 4)

[0249] reverse primer: TGAGCCTCGTGTATCATCTCAAG (SEQ ID NO: 5) and

[0250] the PCR probe was: FAM-TCCACGCCATGTTCCAGCCCT-TAMRA (SEQ ID NO: 6)where FAM (PE-Applied Biosystems, Foster City, Calif.) is thefluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City,Calif.) is the quencher dye.

[0251] For human GAPDH the PCR primers were:

[0252] forward primer: CAACGGATTTGGTCGTATTGG (SEQ ID NO: 7)

[0253] reverse primer: GGCAACAATATCCACTTTACCAGAGT (SEQ ID NO: 8)

[0254] and the PCR probe was: 5′ JOE-CGCCTGGTCACCAGGGCTGCT- TAMRA 3′(SEQ ID NO: 9) where JOE (PE-Applied Biosystems, Foster City, Calif.) isthe fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, FosterCity, Calif.) is the quencher dye.

Example 14

[0255] Northern Blot Analysis Of Clusterin mRNA Levels

[0256] Eighteen hours after antisense treatment, cell monolayers werewashed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then robedusing QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.)using manufacturer's recommendations for stringent conditions.

[0257] To detect human clusterin, a human clusterin specific probe wasprepared by PCR using the forward primer TCCGTACGAGCCCCTGAA (SEQ ID NO:4) and the reverse primer TGAGCCTCGTGTATCATCTCAAG (SEQ ID NO: 5). Tonormalize for variations in loading and transfer efficiency membraneswere stripped and probed for human glyceraldehyde-3-phosphatedehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0258] Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics,Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreatedcontrols.

Example 15

[0259] Antisense Inhibition Of Human Clusterin Expression By ChimericPhosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

[0260] In accordance with the present invention, a series ofoligonucleotides were designed to target different regions of the humanclusterin RNA, using published sequences (GenBank accession numberM64722, incorporated herein as SEQ ID NO: 3, GenBank accession numberL00974, incorporated herein as SEQ ID NO: 10, GenBank accession numberM63377, incorporated herein as SEQ ID NO: 11, GenBank accession numberM63376, incorporated herein as SEQ ID NO: 12, and GenBank accessionnumber M25915, incorporated herein as SEQ ID NO: 13). Theoligonucleotides are shown in Table 1. “Target site” indicates the first(5′-most) nucleotide number on the particular target sequence to whichthe oligonucleotide binds. All compounds in Table 1 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect on humanclusterin mRNA levels by quantitative real-time PCR as described inother examples herein. Data are averages from two experiments. Ifpresent, “N.D.” indicates “no data”. TABLE 1 Inhibition of humanclusterin rnRNA levels by chimeric phosphorothioate oligonucleotideshaving 2’-MOE wings and a deoxy gap TARGET SEQ ID TARGET SEQ ID ISIS #REGION NO SITE SEQUENCE % INHIB NO 129045 5′ UTR 3 18gtctttgcacgcctcggtca 64 14 129046 5′ UTR 3 26 attctggagtctttgcacgc 64 15129047 Start 3 44 gtcttcatcatgcctccaat 68 16 Codon 129048 Coding 3 82tctcccaggtcagcagcagc 67 17 129049 Coding 3 106 tctggtcccccaggacctgc 4618 129050 Coding 3 127 ggagctcattgtctgagacc 59 19 129052 Coding 3 154acttacttccctgattggac 77 20 129053 Coding 3 171 aatttccttattgacgtact 6821 129054 Coding 3 206 gtctttatctgtttcacccc 85 22 129055 Coding 3 286gggcatcctctttcttcttc 64 23 129056 Coding 3 291 atttagggcatcctctttct 3124 129057 Coding 3 303 ttccctggtctcatttaggg 68 25 129058 Coding 3 312tgtctctgattccctggtct 79 26 129059 Coding 3 329 gggagctccttcagctttgt 4927 129060 Coding 3 364 cccagagggccatcatggtc 45 28 129061 Coding 3 369ctcttcccagagggccatca 36 29 129062 Coding 3 385 tcaggcagggcttacactct 7030 129063 Coding 3 412 gtgcgtagaacttcatgcag 60 31 129064 Coding 3 448ggcggccaaccaggcctgag 42 32 129065 Coding 3 449 tggcggccaaccaggcctga 3233 129066 Coding 3 460 actcctcaagctggcggcca 67 34 129067 Coding 3 487agtagaagggcgagctctgg 63 35 129068 Coding 3 497 ttcatccagaagtagaaggg 4136 129069 Coding 3 522 cagcagggagtcgatgcggt 60 37 129070 Coding 3 538gctgccggtcgttctccagc 51 38 129071 Coding 3 556 catccagcatgtgcgtctgc 6939 129072 Coding 3 558 gacatccagcatgtgcgtct 55 40 129073 Coding 3 570gtggtcctgcatgacatcca 62 41 129074 Coding 3 572 aagtggtcctgcatgacatc 4142 129075 Coding 3 609 ctggaagagctcgtctatga 67 43 129076 Coding 3 613tgtcctggaagagctcgtct 69 44 129077 Coding 3 618 gaacctgtcctggaagagct 6845 129078 Coding 3 695 ggaaagaagaagtgaggcct 44 46 129079 Coding 3 726gggcatcaagctgcggacga 65 47 129080 Coding 3 780 ctcaaggaagggctggaaca 8148 129081 Coding 3 781 tctcaaggaagggctggaac 81 49 129082 Coding 3 788tgtatcatctcaaggaaggg 38 50 129083 Coding 3 825 gctgtggaagtggatgtcca 4851 129084 Coding 3 853 attctgttggcgggtgctgg 50 52 129085 Coding 3 858tatgaattctgttggcgggt 36 53 129086 Coding 3 898 ggatctcccggcacacagtc 6854 129087 Coding 3 899 cggatctcccggcacacagt 70 55 129088 Coding 3 911gtggagttgtggvggatctc 69 56 129089 Coding 3 933 gtccttcatccgcaggcagc 5657 129090 Coding 3 972 acagtccacagacaagatct 49 58 129092 Coding 3 1014gagctcccgccgcagcttag 22 59 129093 Coding 3 1027 ggagggattcgtcgagctcc 5560 129094 Coding 3 1088 atcttccactggtaggactt 50 61 129095 Coding 3 1096tgttgagcatcttccactgg 62 62 129096 Coding 3 1118 agctgctccagcaaggagga 4663 129097 Coding 3 1126 gctcgttcagctgctccagc 43 64 129098 Coding 3 1153ttgccagccgggacacccag 72 65 129099 Coding 3 1187 cgcagatagtactggtcttc 6166 129100 Coding 3 1199 accgtggtgacccgcagata 73 67 129101 Coding 3 1221cgagtcagaagtgtgggaag 24 68 129102 Coding 3 1280 gtgatgggatcagagtcaaa 3869 129103 Coding 3 1305 ggagacttctacagggaccg 63 70 129104 Coding 3 1337gccacggtctccataaattt 70 71 129106 3′ UTR 3 1403 gcaaaagcaacatccacatc 7472 129107 3′ UTR 3 1550 tagagtgcaggatccagagc 71 73 129108 3′ UTR 3 1605attagttgcatgcaggagca 71 74 129109 3′ UTR 3 1620 agacagttttattgaattag 1175 129118 Intron 10 2819 cgagatagagccactgtacg 44 76 129119 Intron 104646 tgccaccacccccgggtgat 13 77 129091 Intron- 10 5849gttgttggtggaacagtcca 40 78 Exon Junction 129120 Intron- 10 7384tgcttaccggtgctttttgc 46 79 Exon Junction 129105 Intron- 10 7600acatctcactcctcccggtg 70 80 Exon Junction 129121 3′ UTR 10 7855gaccctccaagcgatcagct 20 81 129122 3′ UTR 10 7863 aaaaagaggaccctccaagC 3982 129115 Intron- 11 322 tgtgtccccttttcacctgg 54 83 Exon Junction 129116Intron 11 445 attaccaatggagcatggca 43 84 129117 Intron 11 810caacatggccaaaccccatg 55 85 129112 Intron 12 1766 gcggcaggtctccaggtctc 4386 129110 Intron 12 4813 ttcccttcggagagtagaga 44 87 129113 Intron 125848 tgcttgggaaatgcctgcaa 34 88 129114 Intron 12 6936agctggatgccagaaaggcc 40 89 129111 5′ UTR 13 39 tggaagtagtggaagccagg 1190

[0261] As shown in Table 1, SEQ ID NOs 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 60, 61, 62, 63, 64, 65, 66, 67, 69, 70, 71, 72, 73, 74, 76, 78, 79,80, 82, 83, 84, 85, 86, 87, 88 and 89 demonstrated at least 30%inhibition of human clusterin expression in this assay and are thereforepreferred. The target sites to which these preferred sequences arecomplementary are herein referred to as “active sites” and are thereforepreferred sites for targeting by compounds of the present invention.

Example 16

[0262] Western Blot Analysis Of Clusterin Protein Levels

[0263] Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to clusterin is used,with a radiolabelled or fluorescently labeled secondary antibodydirected against the primary antibody species. Bands are visualizedusing a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

1 90 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1 tccgtcatcgctcctcaggg 20 2 20 DNA Artificial Sequence Antisense Oligonucleotide 2atgcattctg cccccaagga 20 3 1648 DNA Homo sapiens CDS (53)...(1402) 3cgcggacagg gtgccgctga ccgaggcgtg caaagactcc agaattggag gc atg atg 58 MetMet 1 aag act ctg ctg ctg ttt gtg ggg ctg ctg ctg acc tgg gag agt ggg106 Lys Thr Leu Leu Leu Phe Val Gly Leu Leu Leu Thr Trp Glu Ser Gly 5 1015 cag gtc ctg ggg gac cag acg gtc tca gac aat gag ctc cag gaa atg 154Gln Val Leu Gly Asp Gln Thr Val Ser Asp Asn Glu Leu Gln Glu Met 20 25 30tcc aat cag gga agt aag tac gtc aat aag gaa att caa aat gct gtc 202 SerAsn Gln Gly Ser Lys Tyr Val Asn Lys Glu Ile Gln Asn Ala Val 35 40 45 50aac ggg gtg aaa cag ata aag act ctc ata gaa aaa aca aac gaa gag 250 AsnGly Val Lys Gln Ile Lys Thr Leu Ile Glu Lys Thr Asn Glu Glu 55 60 65 cgcaag aca ctg ctc agc aac cta gaa gaa gcc aag aag aag aaa gag 298 Arg LysThr Leu Leu Ser Asn Leu Glu Glu Ala Lys Lys Lys Lys Glu 70 75 80 gat gcccta aat gag acc agg gaa tca gag aca aag ctg aag gag ctc 346 Asp Ala LeuAsn Glu Thr Arg Glu Ser Glu Thr Lys Leu Lys Glu Leu 85 90 95 cca gga gtgtgc aat gag acc atg atg gcc ctc tgg gaa gag tgt aag 394 Pro Gly Val CysAsn Glu Thr Met Met Ala Leu Trp Glu Glu Cys Lys 100 105 110 ccc tgc ctgaaa cag acc tgc atg aag ttc tac gca cgc gtc tgc aga 442 Pro Cys Leu LysGln Thr Cys Met Lys Phe Tyr Ala Arg Val Cys Arg 115 120 125 130 agt ggctca ggc ctg gtt ggc cgc cag ctt gag gag ttc ctg aac cag 490 Ser Gly SerGly Leu Val Gly Arg Gln Leu Glu Glu Phe Leu Asn Gln 135 140 145 agc tcgccc ttc tac ttc tgg atg aat ggt gac cgc atc gac tcc ctg 538 Ser Ser ProPhe Tyr Phe Trp Met Asn Gly Asp Arg Ile Asp Ser Leu 150 155 160 ctg gagaac gac cgg cag cag acg cac atg ctg gat gtc atg cag gac 586 Leu Glu AsnAsp Arg Gln Gln Thr His Met Leu Asp Val Met Gln Asp 165 170 175 cac ttcagc cgc gcg tcc agc atc ata gac gag ctc ttc cag gac agg 634 His Phe SerArg Ala Ser Ser Ile Ile Asp Glu Leu Phe Gln Asp Arg 180 185 190 ttc ttcacc cgg gag ccc cag gat acc tac cac tac ctg ccc ttc agc 682 Phe Phe ThrArg Glu Pro Gln Asp Thr Tyr His Tyr Leu Pro Phe Ser 195 200 205 210 ctgccc cac cgg agg cct cac ttc ttc ttt ccc aag tcc cgc atc gtc 730 Leu ProHis Arg Arg Pro His Phe Phe Phe Pro Lys Ser Arg Ile Val 215 220 225 cgcagc ttg atg ccc ttc tct ccg tac gag ccc ctg aac ttc cac gcc 778 Arg SerLeu Met Pro Phe Ser Pro Tyr Glu Pro Leu Asn Phe His Ala 230 235 240 atgttc cag ccc ttc ctt gag atg ata cac gag gct cag cag gcc atg 826 Met PheGln Pro Phe Leu Glu Met Ile His Glu Ala Gln Gln Ala Met 245 250 255 gacatc cac ttc cac agc ccg gcc ttc cag cac ccg cca aca gaa ttc 874 Asp IleHis Phe His Ser Pro Ala Phe Gln His Pro Pro Thr Glu Phe 260 265 270 atacga gaa ggc gac gat gac cgg act gtg tgc cgg gag atc cgc cac 922 Ile ArgGlu Gly Asp Asp Asp Arg Thr Val Cys Arg Glu Ile Arg His 275 280 285 290aac tcc acg ggc tgc ctg cgg atg aag gac cag tgt gac aag tgc cgg 970 AsnSer Thr Gly Cys Leu Arg Met Lys Asp Gln Cys Asp Lys Cys Arg 295 300 305gag atc ttg tct gtg gac tgt tcc acc aac aac ccc tcc cag gct aag 1018 GluIle Leu Ser Val Asp Cys Ser Thr Asn Asn Pro Ser Gln Ala Lys 310 315 320ctg cgg cgg gag ctc gac gaa tcc ctc cag gtc gct gag agg ttg acc 1066 LeuArg Arg Glu Leu Asp Glu Ser Leu Gln Val Ala Glu Arg Leu Thr 325 330 335agg aaa tac aac gag ctg cta aag tcc tac cag tgg aag atg ctc aac 1114 ArgLys Tyr Asn Glu Leu Leu Lys Ser Tyr Gln Trp Lys Met Leu Asn 340 345 350acc tcc tcc ttg ctg gag cag ctg aac gag cag ttt aac tgg gtg tcc 1162 ThrSer Ser Leu Leu Glu Gln Leu Asn Glu Gln Phe Asn Trp Val Ser 355 360 365370 cgg ctg gca aac ctc acg caa ggc gaa gac cag tac tat ctg cgg gtc 1210Arg Leu Ala Asn Leu Thr Gln Gly Glu Asp Gln Tyr Tyr Leu Arg Val 375 380385 acc acg gtg gct tcc cac act tct gac tcg gac gtt cct tcc ggt gtc 1258Thr Thr Val Ala Ser His Thr Ser Asp Ser Asp Val Pro Ser Gly Val 390 395400 act gag gtg gtc gtg aag ctc ttt gac tct gat ccc atc act gtg acg 1306Thr Glu Val Val Val Lys Leu Phe Asp Ser Asp Pro Ile Thr Val Thr 405 410415 gtc cct gta gaa gtc tcc agg aag aac cct aaa ttt atg gag acc gtg 1354Val Pro Val Glu Val Ser Arg Lys Asn Pro Lys Phe Met Glu Thr Val 420 425430 gcg gag aaa gcg ctg cag gaa tac cgc aaa aag cac cgg gag gag tga 1402Ala Glu Lys Ala Leu Gln Glu Tyr Arg Lys Lys His Arg Glu Glu 435 440 445gatgtggatg ttgcttttgc accttacggg ggcatcttga gtccagctcc ccccaagatg 1462agctgcagcc ccccagagag agctctgcac gtcaccaagt aaccaggccc cagcctccag 1522gcccccaact ccgcccagcc tctccccgct ctggatcctg cactctaaca ctcgactctg 1582ctgctcatgg gaagaacaga attgctcctg catgcaacta attcaataaa actgtcttgt 1642gagctg 1648 4 18 DNA Artificial Sequence PCR Primer 4 tccgtacgagcccctgaa 18 5 23 DNA Artificial Sequence PCR Primer 5 tgagcctcgtgtatcatctc aag 23 6 21 DNA Artificial Sequence PCR Probe 6 tccacgccatgttccagccc t 21 7 21 DNA Artificial Sequence PCR Primer 7 caacggatttggtcgtattg g 21 8 26 DNA Artificial Sequence PCR Primer 8 ggcaacaatatccactttac cagagt 26 9 21 DNA Artificial Sequence PCR Probe 9 cgcctggtcaccagggctgc t 21 10 8133 DNA Homo sapiens 10 gccatgttgc ccaggctggtctcaaactcc taagctcaag taatcctcct accttggcct 60 cccaaattgt tgggattatagatgtgtgcc actatgccca gccaatgtaa gattttgtag 120 tatattagtg ttgctcctgtcctctgctgc agggcttttt tgattgggac tcagtgaatt 180 gctccaatcc ctgaagtcacatcagttggc ccttagccga gcgggggtgg atatcattgg 240 tggccaaaga tgacagtgaatgaacctgaa atgttgggcc ttgtgacttt tgggcctcca 300 ggtgtctcaa aactgtcccccatggaggga gataaaagga aagagcatgg acctgacaga 360 tggggtgctg ggggctggtcccagctgggc tgttggtcac ttgctgtgtg actgttacag 420 ccatgggcag ggcctggcctggctcaccag ggggtgggag gccaggaggc cgtggccttg 480 gtgagcttct cctaactgtgcccatgctgg ctgtcccagc ttgaggagtt cctgaaccag 540 agctcgccct tctacttctggatgaatggt gaccgcatcg actccctgct ggagaacgac 600 cggcagcaga cgcacatgctggatgtcatg caggaccact tcagccgcgc gtccagcatc 660 atagacgagc tcttccaggacaggttcttc acccgggagc cccaggatac ctaccactac 720 ctgcccttca gcctgccccaccggaggcct cacttcttct ttcccaagtc ccgcatcgtc 780 cgcagcttga tgcccttctctccgtacgag cccctgaact tccacgccat gttccagccc 840 ttccttgaga tgatacacgaggctcagcag gccatggaca tccacttcca cagcccggcc 900 ttccagcacc cgccaacagaattcatacga ggtgagaagg ggtggaagct catggccttt 960 tgagcaactc gttagatgctgagaaccatg ccgagggctc agcgggtgtc atctcgattt 1020 ttctccagca atatcacaagggtgatatta tccttattta aagaggaaaa aaactgagct 1080 gggcatggtg gctcatgcctgtgatgccag cactttgaga ggccaaggcg ggaggatcat 1140 ttgaggccag gagtttgagaccagcctggc caagatagtg agaccctgtc tctacaaaaa 1200 taaaaactta aaaaattagccgggtgtggt ggtgcacacc tgtagtctca gctactcggg 1260 aggctgaggc aagagagtcacctgagcctg gaagttggag gctgcagtga gctatgattg 1320 caccattgca ttccagcctgggcaacagag tgagaccctg tctctaaatt aaaaaataaa 1380 taaaaataac aataggaatcagtggagtcc atctctgcat ggctggatga ctgactcttc 1440 ttccctcgtg tgtccccagaaggcgacgat gaccggactg tgtgccggga gatccgccac 1500 aactccacgg gctgcctgcggatgaaggac cagtgtgaca agtgccggga gatcttgtct 1560 gtgggtgagt cggggtccagaccacaagcc gtcccccctg atcccttgtg tcctggggtc 1620 actggggcct cactggtgctgcctttatgg agtcagacag ataagcgttt ggattccagc 1680 tctgcagcct ttgagctgtgtcccggggca ggtcctgagc ctcatgcagc ttcggttcct 1740 catcttagaa tgagatgatgatgcgaggct gtccctgaag tcggtgagat gtcgttagag 1800 atgcaaaagt gccctccacctggtcggccc catgttgaaa aaagcttgtt gaaaaaagtc 1860 atccccctgg gactccccggtgattctgtt cccaagcgcc aagcagtagg catcttcatt 1920 ttcctctgca gattatgacattgcagacag tatgtgtttt gtttaacaaa actgaccaga 1980 ggccaggcac tgttctaaacactcgacata catttcctca tttcctcaga atgaccctct 2040 gaggaaactg agccacagaaaggttaataa cttatccaag attgaccccg acatgggcga 2100 gctgggcttc aatcctagggcgctgtgttc tctcctgggg cccctcgcag cctctgccac 2160 agaagtcacg ggtctcagtacctgggcatc caagcaatag tccctttggt cggttggttg 2220 gtcccctagg caaagggaatatttcccttt aactgtcccc ctccgtttca ccagctctgg 2280 ttatgggtta acttctttccacttagagat aacagctgtg acagtatttg gactagttcc 2340 tggtacacag cagttcatactcacaaagag ttaattgttt ccccttgttc aacagcttat 2400 cgatctggtg gctttgctcttacttaatgc ttagtttgag tttgccatgg caggccgcca 2460 gggtctagtt aaacattcctagcctcactc ctataatttt agaagccact gcaaaataaa 2520 cagttgtgct ttaacaggctgaagtataag ttgctgtaga tgagtgcaca accaggcctt 2580 ggggcttttt ctataaaaaatatcatagag tggcatcaat tacatggtac ctcaccacaa 2640 gaaagtcatg ttagggtctgagaaaagatg tcagatgcct gtgcccagat tggacctctt 2700 atagctgatt tttactctgttgcccaggct gggtcaggtc tggcccaatc ttaacagtca 2760 ttgattacag ttgagagtgcagccagcgcc agtcttatca gtcattgatt atagctggcg 2820 tacagtggct ctatctcggctcactgcgac ctccgcctcc tgggttcaag tgattctcct 2880 gcctcagcct cccaagtagctgggagtgca ggtgtgcacc accacaccca gctaattttt 2940 gtatttttag tagagacagcatttcactat gttggccagg ctggtcttga actcctgacc 3000 tcaagtaatc tccccgcctcggcctcccaa agttctggga ttacaggtgt gagccactgt 3060 gcctgacctg agatagattcttagagaatt attggtaaga ataattctct aagctgagct 3120 aaatagtcta cactgaagaggactgcctac tgttatttaa ggtgcttgca accatataag 3180 catgtactgc ctgggaactctagatgagga tttctcaatt tcagcgctgt tgattttttt 3240 tttttttttt gagacagggtctctctctat cacccagcct ggagtgcagt ggcaccatta 3300 cagctcactg cagcctagacctcttgggct gaagtcatcc tcctgcctca gcctcctgag 3360 taacagacta caggtgtgctccaccatgct tggctaattt ttttattttt agtagagatg 3420 gggtcttgct acattgcccaagctggtctc taactcctgg gctcaagtga tcctcctacc 3480 tcagcctccc agagtgctgggattacaggt gtgagcagtg ctgacatttt ggaccaggtc 3540 attctttgtc gttgggggctgtcctgagca gttcagggtg tttggcagca ttcctggcct 3600 ctgcccacta gaggtcagcagctcccttcc ctttgttgtg acaaccagct tcagaacttg 3660 ctaaatctcc ctgggtgacagcgtccacag tagagaacct ctattctaga ctaagcctca 3720 gctcttaagg atttttcttattttattatt atttttttaa gacagggtct cgctctatca 3780 cccaggctgg agcgtagtggcgcaatcttt gctcactgca acctctgctt cctgggttca 3840 agcgatttct cctgccccagcctcctgagt agctgggatt acaggcgtgc accgccacgc 3900 ctggctaatt tttatatttttagtagagac agggtttcac catattggcc aggctggtct 3960 caaactcttg acctcaagtgatcagcctgc ctcagcctcc caaagtgctg ggattacagg 4020 tgtgagccag cacgcctggctagtttttct tatttttaaa tttttttttg gtaaaataat 4080 gatgtttatt tattacatatttattttcaa actggcatct tgttagtaat tctgtttctt 4140 tccccaccta acattttgtttactataaat gatttcagtc atcatcctaa agcatatgca 4200 aaatctccct tcccctgactcacgtttgat gtacctgcct ctggatattt ttgaaatacc 4260 ttagggggag aaaaacagtagttttaagag ctagtggaca gtttccaggt cttaatgaat 4320 ctgacaacct gcagcccagggccaagagga atgaattctc ttttccctgc tctcttgatg 4380 aactcactga ccagccatgggcggcaggtg ggcaggcaag gacccctggc caccaggtgc 4440 cagtgcatca gctgcatgaactcctggcac cagaactgcc acctctacag acatgctcaa 4500 aagacaagtt tggaccgggtgcattggctc acacctgtaa tcccagcacc ttgagaggcc 4560 gaggtgggtg gacccctgaggtcaggagtt tgagaccagc ctagccaaca tggtgaaacc 4620 ctgtctctcc taaaaatacaaaaaaatcac ccgggggtgg tggcaggcac ctgtaatccc 4680 aactactctg gaggctgaggcaggagaatt gcttgaaccc gggaggtgga ggttgcagtg 4740 agctgagctc gcgccattgcactccagcct gggaaacaag agcgaaattc tgtctcaaaa 4800 aaaagacaag cttggaggattgtccagaac cacagatcca gggtaggaaa agcccaagct 4860 taggagctga agaccctggttcaatcccgg gcccagagat catttattct atggctttag 4920 gtaagctatt tattgatacttctgtgggcc tcagtttcat tattggtaaa aattatttca 4980 ttattggtaa aattaggacttaagtcctaa tccttaagtc agaacagatc caattcttag 5040 agaaaaagga tatccagagagaactttctg cggtgtctgg gacgcaggca gtgccacacg 5100 aatggcagct gtgagtaatattcctcctct ctggaaatga ttcccgggag gactagggca 5160 acgagagcca ctccaggtctgagaacatgg agaacttgag atcagtgctt ttggaagtgt 5220 ggtcaacaca gtttgtcaccaaagagataa gggtctggca cccaaagata aatgaatgat 5280 gttacgaagc acactgtttaggtcagttgg cgtatttttc cagagcaagg cttctcaggc 5340 tgggcgtggt ggctcacaccagtaatccca gcactttttg ggcagatggg ttgagcccag 5400 gagttcgaga ccagcctggacaacacagag aaaccccgtg tctacaaaaa atacaaaaat 5460 tagctgggca tggtagcatgtgcctatagt cccagctact caggaggctg aggttggagg 5520 acagcctgag cctgggaagtcaaggctgca gtgagccgag atctcaccac tgtattccag 5580 cctaggcaac agagcaaaactctgtctcaa aaaaacaaaa acaaaaacaa aaaacccaaa 5640 agactttctg gatgacggaagcagtgtcta gattcacatt ctgaggcaaa acctttattt 5700 tgtcgtggac aattccagtttgtggccctt cccttaggga agcactgctt ttgttcccgc 5760 tgcatgtgct aacttccattcattcatggt tctatccctt tgtagccttc ccttcacact 5820 tctcacttgc gtttcttccatctctgggca gactgttcca ccaacaaccc ctcccaggct 5880 aagctgcggc gggagctcgacgaatccctc caggtcgctg agaggttgac caggaaatac 5940 aacgagctgc taaagtcctaccagtggaag atgctcaaca cctcctcctt gctggagcag 6000 ctgaacgagc agtttaactgggtgtcccgg ctggcaaacc tcacgcaagg cgaagaccag 6060 tactatctgc gggtcaccacggtgagctgt gtcccggcca catgctgtgg ctcgggagcc 6120 gagctgtgat cgggagcaggggcatgtgtg cttttgactg agcatttatc acacggcaga 6180 aaatagaaaa ctttaggcgcccctgttgcc ttgaagcctc atcacccact cagggaaaat 6240 ataaccctgc tttacaaaggagcaaagtaa gagaggttcc acagcttggc caaggtgtga 6300 tagctgacag atgacttggacgggtatttg aacctgactg cctggctgcc aagcctgtat 6360 tttgttgttg ttgtttttgttttggtgcac aaatctgtga ataaaccaga agcctctgtt 6420 cttttctcaa agctacaaggctgccctctg gcatgtaaaa tggcttatga attagtacat 6480 cactctctgc cagtgataaaaacttctctc taggccagac atggtggctc atgcctgtaa 6540 tcccagcact ttgggaggcagaggcaagag gattgcttga ggccaggaat ttgagaccag 6600 cctgggcaac acagcaagattccctctcta caaaaaatac aaaaatcagt caggtgtggt 6660 ggcacacact tgtagtcccagctattcagg aggctgaggt gggaggattg cctgagccct 6720 gaagtggagg ctgcagtgagctgtgatcac gccactgcac tccagcctgg gtgacagagt 6780 gagactctgt ctcttaaaaaatatatatat ataaaataat aaaataaagt taaaaaatca 6840 aataaaactt atttctagtactgggaactc ttctttttct tttctttctt ccctccaggc 6900 cctctggatt ccttttctaccctactctga ccaagggctg cctaaagcaa atgtttggaa 6960 accactttta ttctttggggtgctccctgg ctggtcattt gcagatgaca tttgccccaa 7020 cacatgagtg tctgtgaaccaggtccgttc tgtccactga gctgtactta cgtctagatg 7080 tataagaagc atggggtcagctctctaggt tccttggagg agcaggagga cttccttatc 7140 agaagcctga cttctgttgcagagcgcatg cattttgacc acagtgtttc agctcttccc 7200 ttttctcttg ttccatttaggtggcttccc acacttctga ctcggacgtt ccttccggtg 7260 tcactgaggt ggtcgtgaagctctttgact ctgatcccat cactgtgacg gtccctgtag 7320 aagtctccag gaagaaccctaaatttatgg agaccgtggc ggagaaagcg ctgcaggaat 7380 accgcaaaaa gcaccggtaagcaggcgggc ctttcctgcg gcctgcaggg cccagtgagt 7440 ctctgggagc cacaaaaaaacaaacaaagt gcagactcta tagcctggtg ggaacgactc 7500 cgcccggagc cagagcccaagaacaaagcc aggaagttac gggggaattt tatttttcct 7560 ttggaggatg ttttactttggaggataact gttttttatt tcagggagga gtgagatgtg 7620 gatgttgctt ttgcacctacgggggcatct gagtccagct ccccccaaga tgagctgcag 7680 ccccccagag agagctctgcacgtcaccaa gtaaaccagg ccccagcctc caggccccca 7740 actccgccca gcctctccccgctctggatc ctgcactcta acactcgact ctgctgctca 7800 tgggaagaac agaattgctcctgcatgcaa ctaattcaat aaaactgtct tgtgagctga 7860 tcgcttggag ggtcctctttttatgttgag ttgctgcttc ccggcatgcc ttcattttgc 7920 tatggggggc aggcaggggggatggaaaat aagtagaaac aaaaaagcag tggctaagat 7980 ggtataggga ctgtcataccagtgaagaat aaaagggtga agaataaaag ggatatgatg 8040 acaaggttga tccacttcaagaattgcttg ctttcaggaa gagagatgtg tttcaacaag 8100 ccaactaaaa tatattgctgcaaatggaag ctt 8133 11 940 DNA Homo sapiens 11 aagcttgaac tggagcaagggtaggcactt gcatgctggg tggccagcct atgggaaggc 60 tcgccctggg gcagagggcctggcacccag cagctctttg agtgcatgag cctgtggtct 120 ctgtgtgctc agccagccttgtgtcttcct gtaggatgcc ctaaatgaga ccagggaatc 180 agagacaaag ctgaaggagctcccaggagt gtgcaatgag accatgatgg ccctctggga 240 agagtgtaag ccctgcctgaaacagacctg catgaagttc tacgcacgcg tctgcagaag 300 tggctcaggc ctggttggccgccaggtgaa aaggggacac atgagtggcc aaggctctga 360 gtggggaagg aggggagcctagtgaaatat gcttcattcc gcatgccaga tgcaattgat 420 tagcattggc tggcttgcccagagtgccat gctccattgg taatgtctgg catgagtaga 480 gagagtggag tcatcaaaaggatgtaggcc aggtatctgc cttctcttag aaaactcatg 540 cagcagtgct tagctggatgacataataaa ctgcttcgtg ggatgcagag ccctgtgtca 600 cttatgtgga aggatttaagaatttttttt tttttttgag acagggtctc actctgtcac 660 ccaggctgga gtacagtgatgtgatcatgt ttcactgcag cttcgacctc ctgggttcag 720 gtgatcctcc cacctcagcctcccaagtag ctgggactac aggcacgtac caccacaccc 780 agctaatttt tgtattttttttttgtaaac atggggtttg gccatgttgc ccaggctggt 840 ctcaaactcc taagctcaagtaatcctcct accttggcct cccaaattgt tgggattata 900 gatgtgtgcc actagtcccagccaatgtaa gattttgtag 940 12 7610 DNA Homo sapiens unsure 5461 unknown12 gacctgcagg tcaacggatc cattcccgat tcctcatcgt ccagatggaa gaaactgagg 60cccaagggca aagtgattag tccgaggtca cccagtgtct aggggcacac ctaggactgt 120aatcagactt tcatggacct ggtctgggtt ctcccactta gtcatgggcc ttgaagattc 180cccgaggctg cctcctgaaa aggactgggg tctagtggcc cctggacgtt gggcaagcaa 240gggactgggc ctccatgttg tgcctccata gtcctgatcc tgaactggaa aactcagccc 300ctgaccacgc agctctcctt taagcccctt tgtttcacat ggttttcaaa gtctgccacc 360cacagtgggg ctgcctgtac ccgccctgtc cacccattgc cccagctgtc agccccttga 420cttctctcct ggggcttaaa catccctggc tccaaaatgg gcagctcact ttcttcccca 480agaagtagct gcacctccag ggttcctaga tttgcccctc cttgccaggg ggaggggtgg 540ctgcgacagg agattctccc tgctctcagc agaaggaact ccagcagttg gagaccagca 600aacccctctg gacacagatc tgatttccta actgggaagg ctcagggcaa aataaaaatt 660caggtccact ggttcaaaaa ctatgaagaa tttcaagacc gtcacagtag cccattaaac 720caaacgtgga tctgcaaggg tcccacagcc atgaagccca ccctgcttgg ttgggttcca 780aaaagatggg gacagtgatt gcttaagctc tgtggatcaa ggaccccgga gaggccttct 840ggctctccac atatctgctc tgatcactcc taaacacaat tctgtttcct ccaggcctgg 900cgggtcagtc cagggacccc catcagtgtg atgtttccag gagtaggcgt ttcaatactt 960cctgtgctct cttctccagc acaaggcccc tctccatccc accctcatta tgtctgactc 1020tttactattt aaatgggtca agagaagtgg cgcttgtgta atgtgaaggt taaggtcagt 1080agggccaggg aactgtgaga ttgtgtcttg gactgggaca gacagccggg ctaaccgcgt 1140gagagggctc ccagatggca cgcgagttca ggctcttccc tactggaagc gccagcgccg 1200cacctcaggg tctctcctgg agccagcaca gctattcgtg gtgatgatgc gcccccccgc 1260gccccagccc ggtgctgcac cggcccccac ctcccggctt ccagaaagct ccccttgctt 1320tccgcggcat tctttgggcg tgagtcatgc aggtttgcag ccagccccaa aggtgtgtgc 1380gcgaacggag cgctataaat acggcgcctc ccagtgccca caacgcggcg tcgccaggag 1440cagcagcatg ggcacagggt ccgtgaccgg tgagatgtcc ccgtcttccc tacccttgag 1500cagagccaca ccaggacgga tgggcgggca ggggatggca gccaggcaga gagggatgac 1560acagctcgca gtcacaaccc ctgcgctttc gacggagccc aggaagccag ggaggggagg 1620tgccggagcc ccatcaccag gcagctgagc caggggccgc gcaaccgccg cctgatgagc 1680acgagcttca cgcaaccaca attctgtggt gggggggtaa atagaacaga tataatgatc 1740atcctttcgc aaagatgggg aaactgagac ctggagacct gccgcgttgg cagacccagg 1800ctagcaggtg acagagctgg cctgcaccga gctccttcct gcagcatatc ctctgcgaag 1860atgcggatct ctcagttgtg gctttcggct tgcatgcatg agtcatctag ttttcttcta 1920aattctctag ctctctggac actgttgcct gtaagtatga ggctgcggat ttcagtatat 1980ctgcaaccac cgaaatccga ctttttctgc ctcctaatgc atctgaggtg catcagagaa 2040aagtcacaca agatccacca ggcctcagac ctctgattcc acagtctcat tttacagatg 2100ataatctgag gcctggagag gtttaggact ggtgccaaca ctaaacagca aataagtatc 2160agaattggga ttcgagccaa agcctcttga ccttccagaa tttctggacc tagttaaaaa 2220aaatatgatt tttattatta ttttttaaac ggagaggtta ggaatttaaa ggaaagtaca 2280gatactatat aaaaaaagat gcccatgaaa atgttaagtt ataataatag tggagcattg 2340ggcacaactg aaatggccaa tcttgtgaga atggtaaaat aaacttaggt ccgtgagtaa 2400gtggagtatt acatagccat aaaagtatgc ccttaaagaa tatttgaaga tggtgaatgt 2460gaagaatctt gtataaactg catggaagac agaaggaaat ataccacagt gctaaccttt 2520gcctctgggt gatatgaatt accggtgatt atttttctta ttttcctttt ggtttagttt 2580tctccatttg aagaagcaga taggagccgg ggctttggga ttgaaaccct caccatctgt 2640gtgccctctt cactgtcttc ccatcctccc cacggctccc tgttcacagt cattgatttt 2700ctttctttct tttctctttt tttttttttt tcctgagacc aagtctcact ctgttgccca 2760ggctggagta gagtagcgcc atctcggctc actgcaacct ccgccatccg ggttcaagca 2820gttctcatgc ctcagcctct gagtagctgg gactacagcc gcatgctgct acatccggct 2880aatttttgta tttttagtag agacatggtt tcaccacctt ggccaggctg gtctcgaact 2940cctgatctca agtaatccag cctgtcttgg cctcccaaag tgctggggtg acaggtgtga 3000atcaatgcgt ccctgccagg tcattgattt tcttaagcct ctagccctgc cctgcttgga 3060aacgttttgg gaagctgctc agttcaaagt tcccaggagg gtgtgcctgg aggggagttg 3120ctcccaaagt ctgcctgctc cccccgcccc ccctgccccc caccccccgc catcttctcc 3180tcctcctctt cccctgagca gcccctttgt ccacagaacc ggccttttct ggtagaagga 3240gcaaggccaa gtggtttaag ccttcttagg gagaatgagg ctgtgtggta gtgctgggga 3300ctcgagggcc ttgcgttggc atggctcttc cacccagggc agctggcagc caggctccca 3360ggaggcagag gagatgaggg gggaggtgag tccgagcaaa ggaaaggagg tcggctgtgc 3420agtcacggtt ctagaacatt cattggatca gcagcatcca tatcacctgc agactggctg 3480gaaaagcagt ctcagaacca acattataac cagccctgca gtgattcata agtactttaa 3540aaagtggtca atcatttcag caaagcagag ccacacagtc cgggggacca caggtggcct 3600ctgtgtgctt gtctcggttt tcctgcccct ctccagacat gttgattaga cactgccaat 3660gcccagcctc agacctcagt ctaatttgga agtagtcaga atttactatg attacataag 3720accctcgtgt ttacagaaca cattcccctc tctgaggtct ggattagatc cattttacag 3780atgaagaaac tgaggctcag atatttaagt gacttggaat caaggaaaga atactggaca 3840tggggctggg agggctgggc tctcatccca gggttaccat gagcatgctg tggactctag 3900ggagtccatg ccctctctgg cgttcagctc accgctaggt agagaggttg ggtgagagaa 3960cgacctcctt cccaggtctg agctggatgg ttcaccaggg accccaggct ccctggacga 4020gactctgtgc ccgctgctga gtctggaatt cctttcctgt atcttgcctt tgcgtgcccc 4080attcttcatg gcccagcacc ctgtcttctg gtcagaacct agttctgaat gggtttttcc 4140agaagttgtt gctttcaggg gcccctggca gagaggtgtt tctggctggc tttgtctctc 4200tggcatgaca aaggctctgt tcctgctgga ggcatttcag ggctcagtgg gcagctgggg 4260cagacgctga gaccacagcc ttcctggtga gcccggtctc cgccccctac cccatctctg 4320ggaaggcgct gaccccatct cttctcccac gctgctccct ggctctttgc gcctgattac 4380ttctcatgag aggcactcct tgttaatgtg ctactgagtg tccagatggg cctgctgggc 4440tgagcgggct ttggatgtga accatttcag gaaggggaac ccatcgtcct gttggttctg 4500tgatggcaaa tgggtgagct cagataacga gttcttggga ggggcatggt gggggtggag 4560tgcaggggga ggggtttctg ttttattgac aacagcctca gcttctggga aagggtccat 4620tgtgtaagac cggggctatg gctgtgcccc gtggctcagg gcagccagcc agtggtggca 4680ggaacactgg cagggcagcc tcgtgtcggc ttagagggga tgggcagtgt ggagggcctg 4740gcagagcaag aggactcatc cttccaaagg gactttctct gggaagcctg ctcctcgggc 4800cactgcgaac cctctctact ctccgaagga attgtccttc ctggcttcca ctacttccac 4860ccctgaatgc acaggcagcc cggcccaagt ctcccactag gatgcagatg gattcggtgt 4920gaagggctgg ctgctgttgc ctccgcgtct tgaaagtcaa gttcaggtgg tgctgagact 4980ccctgggggc tgcagcgctg tggtgaatgg ggagcgtctg ctggggtgaa ggtttaggtg 5040cacattgcag aggacgtggc tggtctctgg gatgcagtcc ctctgtggag gtggcatggg 5100gagggacgga tgcatgacct aagggtggta ttttcagtgt ctgacatgat cgataccact 5160ctggacaagg aggccaggat gcagaaagcc tgtgtgcctc gctgattgtc ggggaggatg 5220tggcttggac aagagcctgg ttcctccgat gccagggttc ttgtttcttc cactcaacat 5280tgctgtcctg cagtccctcc ctccctgcac ctcctgcctt cgctttcatt cgaggtgtcc 5340atggcaagtc tggtcatttc cccccatttc ctcaggaata aaagttgcag cagtgcctgc 5400tgtggggaca gctgagggca gtgaggctgg ggagctgctg cagggcggag tgggcgggac 5460nnagcaggct gtctagctgt tcccatgatg gtctcctgtt ctctgcagag gcgtgcaaag 5520actccagaat tggaggcatg atgaagactc tgctgctgtt tgtggggctg ctgctgacct 5580gggagagtgg gcaggtcctg ggggaccaga cggtctcaga caatgagctc cagggtgagt 5640agaccaagca tgatgttcct ctggccacag ggtgatgagg tcagagggca gggtagctaa 5700ttctgctcag tgcctctcta tcaggcccca gtgttacaga ccgtttttat cttgtgcact 5760gggtctgggt gcctgtgtct gggcccactc tgagcctcag ctcccaggcc cctggttcag 5820gctctgcgtg catcagactg ccggcatttg caggcatttc ccaagcactt tcggctgttg 5880catttcattc agctcttccc ctcccaggcc ccttagccca gctcccaggc ctcctccaca 5940aagctgtgtc tggaccaccg gagctcttat ccctctcccc tttggagtgc ccagagctta 6000tccctcctgt gagctgacgg tttctgcagg atcattgtta aaaacccaga tcagacatgg 6060gtgtgagtct gtttcacctc ttctcagctg ggtgactttg ggccactatc ttgatctcat 6120gacactcccc ccacccccca ttttattgag atataattaa caaataaaaa ttgtgtatat 6180ttaaggtata tgacgtgatg ttttgaaatg cacatacatt gaaatgatga ccagttttta 6240tggtgggacg gtgggaagac ttaaaatcta ctttcttagc aaatttccag ttatgatatg 6300gtgttattaa ctataagcac cacctgtatg ttagacctcc agaacatact cctcctacct 6360gatgaacact ttgacccttt atcatatcac acttcccatg tctccctctg cgaagtgggc 6420acggcggggg gctggagcat tacgtaaact gcacatgaag tgtttggcgc agtgcttggc 6480atgggataaa caccagtgaa gtagcactta ggtgacacag tgtttcgctg catttgtcac 6540cagtgctatc cttactcatt tactcatctt cttattcctg tcgcctggca ctgcattgga 6600acaaagaaat acacatatct gtttaaactg aactctagaa agatttgtgt ccaaaataac 6660aatattttat attttgatgc tgcaaacgtg acacttctgg gttttttttt tttccttgcc 6720aagtttcttc tgcacccagc tcattctcca ggggcacatg gcagtggctg ggcataactc 6780tgggtgtgcc ggctcccatg gtctgcattt ctaagcagta gggtgcagtc agcaaggagc 6840ctgtgatggg agcctgtgcc agggcaaggc tggggcatgc tgctgcctgc tggcaggagt 6900gggggtccca gccttgacag cccctgaact gaacgggcct ttctggcatc cagctcattc 6960cagggtcctg aggccacctc ttcctctcgc ctcattctgc ctcttgcact tctcttgcag 7020aaatgtccaa tcagggaagt aagtacgtca ataaggaaat tcaaaatgct gtcaacgggg 7080tgaaacagat aaagactctc atagaaaaaa caaacgaaga gcgcaagaca ctgctcagca 7140acctagaaga agccaagaag aagaaagagg tcaggaggag ccgctaccgc ctccctgcct 7200tgaccatccc actggagggg agggaggggg tcactgcgcg gtgccctgct ggttgccatg 7260gtgacccgca gtcctcccag gctgtgtcag ctgatgctga ggctgcagtt aagaagcagg 7320gaaggttcat ttgcttctga aagcatcagg gagtgagatc ttggatctgg ttttgttatg 7380agcctggccc agggctaatg ccagattcat ttcaatagat gtttctaagc cctgatcacg 7440tgctagttcc aagcaggctc tgggtggggt ggcggcaggg gccagacagg cgtggcgtcc 7500aaccttcagg aagcttctag gagttaggga acagttggat cttgaaggat gagtgggttc 7560tttaagccag gtgggaaggg gattccaggt gggcgaatga ggggaagctt 7610 13 1651 DNAHomo sapiens CDS (199)...(1545) 13 ctgcgaaccc tctctactct ccgaagggaattgtccttcc tggcttccac tacttccacc 60 cctgaatgca caggcagccc ggcccaagtctcccactagg gatgcagatg gattcggtgt 120 gaagggctgg ctgctgttgc ctccggctcttgaaagtcaa gttcagaggc gtgcaaagac 180 tccagaattg gaggcatg atg aag act ctgctg ctg ttt gtg ggg ctg ctg 231 Met Lys Thr Leu Leu Leu Phe Val Gly LeuLeu 1 5 10 ctg acc tgg gag agt ggg cag gtc ctg ggg gac cag acg gtc tcagac 279 Leu Thr Trp Glu Ser Gly Gln Val Leu Gly Asp Gln Thr Val Ser Asp15 20 25 aat gag ctc cag gaa atg tcc aat cag gga agt aag tac gtc aat aag327 Asn Glu Leu Gln Glu Met Ser Asn Gln Gly Ser Lys Tyr Val Asn Lys 3035 40 gaa att caa aat gct gtc aac ggg gtg aaa cag ata aag act ctc ata375 Glu Ile Gln Asn Ala Val Asn Gly Val Lys Gln Ile Lys Thr Leu Ile 4550 55 gaa aaa aca aac gaa gag cgc aag aca ctg ctc agc aac cta gaa gaa423 Glu Lys Thr Asn Glu Glu Arg Lys Thr Leu Leu Ser Asn Leu Glu Glu 6065 70 75 gcc aag aag aag aaa gag gat gcc cta aat gag acc agg gaa tca gag471 Ala Lys Lys Lys Lys Glu Asp Ala Leu Asn Glu Thr Arg Glu Ser Glu 8085 90 aca aag ctg aag gag ctc cca gga gtg tgc aat gag acc atg atg gcc519 Thr Lys Leu Lys Glu Leu Pro Gly Val Cys Asn Glu Thr Met Met Ala 95100 105 ctc tgg gaa gag tgt aag ccc tgc ctg aaa cag acc tgc atg aag ttc567 Leu Trp Glu Glu Cys Lys Pro Cys Leu Lys Gln Thr Cys Met Lys Phe 110115 120 tac gca cgc gtc tgc aga agt ggc tca ggc ctg gtt ggc cgc cag ctt615 Tyr Ala Arg Val Cys Arg Ser Gly Ser Gly Leu Val Gly Arg Gln Leu 125130 135 gag gag ttc ctg aac cag agc tcg ccc ttc tac ttc tgg atg aat ggt663 Glu Glu Phe Leu Asn Gln Ser Ser Pro Phe Tyr Phe Trp Met Asn Gly 140145 150 155 gac cgc atc gac tcc ctg ctg gag aac gac cgg cag cag acg cacatg 711 Asp Arg Ile Asp Ser Leu Leu Glu Asn Asp Arg Gln Gln Thr His Met160 165 170 ctg gat gtc atg cag gac cac ttc agc cgc gcg tcc agc atc atagac 759 Leu Asp Val Met Gln Asp His Phe Ser Arg Ala Ser Ser Ile Ile Asp175 180 185 gag ctc ttc cag gac agg ttc ttc acc cgg gag ccc cag gat acctac 807 Glu Leu Phe Gln Asp Arg Phe Phe Thr Arg Glu Pro Gln Asp Thr Tyr190 195 200 cac tac ctg ccc ttc agc ctg ccc cac cgg agg cct cac ttc ttcttt 855 His Tyr Leu Pro Phe Ser Leu Pro His Arg Arg Pro His Phe Phe Phe205 210 215 ccc aag tcc cgc atc gtc cgc agc ttg atg ccc ttc tct ccg tacgag 903 Pro Lys Ser Arg Ile Val Arg Ser Leu Met Pro Phe Ser Pro Tyr Glu220 225 230 235 ccc ctg aac ttc cac gcc atg ttc cag ccc ttc ctt gag atgata cac 951 Pro Leu Asn Phe His Ala Met Phe Gln Pro Phe Leu Glu Met IleHis 240 245 250 gag gct cag cag gcc atg gac atc cac ttc cac agc ccg gccttc cag 999 Glu Ala Gln Gln Ala Met Asp Ile His Phe His Ser Pro Ala PheGln 255 260 265 cac ccg cca aca gaa ttc ata cga gaa ggc gac gat gac cggact gtg 1047 His Pro Pro Thr Glu Phe Ile Arg Glu Gly Asp Asp Asp Arg ThrVal 270 275 280 tgc cgg gag atc cgc cac aac tcc acg ggc tgc ctg cgg atgaag gac 1095 Cys Arg Glu Ile Arg His Asn Ser Thr Gly Cys Leu Arg Met LysAsp 285 290 295 cag tgt gac aag tgc cgg gag atc ttg tct gtg gac tgt tccacc aac 1143 Gln Cys Asp Lys Cys Arg Glu Ile Leu Ser Val Asp Cys Ser ThrAsn 300 305 310 315 aac ccc tcc cag gct aag ctg cgg cgg gag ctc gac gaatcc ctc cag 1191 Asn Pro Ser Gln Ala Lys Leu Arg Arg Glu Leu Asp Glu SerLeu Gln 320 325 330 gtc gct gag agg ttg acc agg aaa tat aac gag ctg ctaaag tcc tac 1239 Val Ala Glu Arg Leu Thr Arg Lys Tyr Asn Glu Leu Leu LysSer Tyr 335 340 345 cag tgg aag atg ctc aac acc tcc tcc ttg ctg gag cagctg aac gag 1287 Gln Trp Lys Met Leu Asn Thr Ser Ser Leu Leu Glu Gln LeuAsn Glu 350 355 360 cag ttt aac tgg gtg tcc cgg ctg gca aac ctc acg caaggc gaa gac 1335 Gln Phe Asn Trp Val Ser Arg Leu Ala Asn Leu Thr Gln GlyGlu Asp 365 370 375 cag tac tat ctg cgg gtc acc acg gtg gct tcc cac acttct gac tcg 1383 Gln Tyr Tyr Leu Arg Val Thr Thr Val Ala Ser His Thr SerAsp Ser 380 385 390 395 gac gtt cct tcc ggt gtc act gag gtg gtc gtg aagctc ttt gac tct 1431 Asp Val Pro Ser Gly Val Thr Glu Val Val Val Lys LeuPhe Asp Ser 400 405 410 gat ccc atc act gtg acg gtc cct gta gaa gtc tccagg aag aac cct 1479 Asp Pro Ile Thr Val Thr Val Pro Val Glu Val Ser ArgLys Asn Pro 415 420 425 aaa ttt atg gag acc gtg gcg gag aaa gcg ctg caggaa tac cgc aaa 1527 Lys Phe Met Glu Thr Val Ala Glu Lys Ala Leu Gln GluTyr Arg Lys 430 435 440 aag cac cgg gag gag tga gatgtggatg ttgcttttgcacctacgggg gcatctgagt 1585 Lys His Arg Glu Glu 445 ccagctcccc ccaagatgagctgcagcccc ccagagagag ctctgcacgt caccaagtaa 1645 ccaggc 1651 14 20 DNAArtificial Sequence Antisense Oligonucleotide 14 gtctttgcac gcctcggtca20 15 20 DNA Artificial Sequence Antisense Oligonucleotide 15 attctggagtctttgcacgc 20 16 20 DNA Artificial Sequence Antisense Oligonucleotide 16gtcttcatca tgcctccaat 20 17 20 DNA Artificial Sequence AntisenseOligonucleotide 17 tctcccaggt cagcagcagc 20 18 20 DNA ArtificialSequence Antisense Oligonucleotide 18 tctggtcccc caggacctgc 20 19 20 DNAArtificial Sequence Antisense Oligonucleotide 19 ggagctcatt gtctgagacc20 20 20 DNA Artificial Sequence Antisense Oligonucleotide 20 acttacttccctgattggac 20 21 20 DNA Artificial Sequence Antisense Oligonucleotide 21aatttcctta ttgacgtact 20 22 20 DNA Artificial Sequence AntisenseOligonucleotide 22 gtctttatct gtttcacccc 20 23 20 DNA ArtificialSequence Antisense Oligonucleotide 23 gggcatcctc tttcttcttc 20 24 20 DNAArtificial Sequence Antisense Oligonucleotide 24 atttagggca tcctctttct20 25 20 DNA Artificial Sequence Antisense Oligonucleotide 25 ttccctggtctcatttaggg 20 26 20 DNA Artificial Sequence Antisense Oligonucleotide 26tgtctctgat tccctggtct 20 27 20 DNA Artificial Sequence AntisenseOligonucleotide 27 gggagctcct tcagctttgt 20 28 20 DNA ArtificialSequence Antisense Oligonucleotide 28 cccagagggc catcatggtc 20 29 20 DNAArtificial Sequence Antisense Oligonucleotide 29 ctcttcccag agggccatca20 30 20 DNA Artificial Sequence Antisense Oligonucleotide 30 tcaggcagggcttacactct 20 31 20 DNA Artificial Sequence Antisense Oligonucleotide 31gtgcgtagaa cttcatgcag 20 32 20 DNA Artificial Sequence AntisenseOligonucleotide 32 ggcggccaac caggcctgag 20 33 20 DNA ArtificialSequence Antisense Oligonucleotide 33 tggcggccaa ccaggcctga 20 34 20 DNAArtificial Sequence Antisense Oligonucleotide 34 actcctcaag ctggcggcca20 35 20 DNA Artificial Sequence Antisense Oligonucleotide 35 agtagaagggcgagctctgg 20 36 20 DNA Artificial Sequence Antisense Oligonucleotide 36ttcatccaga agtagaaggg 20 37 20 DNA Artificial Sequence AntisenseOligonucleotide 37 cagcagggag tcgatgcggt 20 38 20 DNA ArtificialSequence Antisense Oligonucleotide 38 gctgccggtc gttctccagc 20 39 20 DNAArtificial Sequence Antisense Oligonucleotide 39 catccagcat gtgcgtctgc20 40 20 DNA Artificial Sequence Antisense Oligonucleotide 40 gacatccagcatgtgcgtct 20 41 20 DNA Artificial Sequence Antisense Oligonucleotide 41gtggtcctgc atgacatcca 20 42 20 DNA Artificial Sequence AntisenseOligonucleotide 42 aagtggtcct gcatgacatc 20 43 20 DNA ArtificialSequence Antisense Oligonucleotide 43 ctggaagagc tcgtctatga 20 44 20 DNAArtificial Sequence Antisense Oligonucleotide 44 tgtcctggaa gagctcgtct20 45 20 DNA Artificial Sequence Antisense Oligonucleotide 45 gaacctgtcctggaagagct 20 46 20 DNA Artificial Sequence Antisense Oligonucleotide 46ggaaagaaga agtgaggcct 20 47 20 DNA Artificial Sequence AntisenseOligonucleotide 47 gggcatcaag ctgcggacga 20 48 20 DNA ArtificialSequence Antisense Oligonucleotide 48 ctcaaggaag ggctggaaca 20 49 20 DNAArtificial Sequence Antisense Oligonucleotide 49 tctcaaggaa gggctggaac20 50 20 DNA Artificial Sequence Antisense Oligonucleotide 50 tgtatcatctcaaggaaggg 20 51 20 DNA Artificial Sequence Antisense Oligonucleotide 51gctgtggaag tggatgtcca 20 52 20 DNA Artificial Sequence AntisenseOligonucleotide 52 attctgttgg cgggtgctgg 20 53 20 DNA ArtificialSequence Antisense Oligonucleotide 53 tatgaattct gttggcgggt 20 54 20 DNAArtificial Sequence Antisense Oligonucleotide 54 ggatctcccg gcacacagtc20 55 20 DNA Artificial Sequence Antisense Oligonucleotide 55 cggatctcccggcacacagt 20 56 20 DNA Artificial Sequence Antisense Oligonucleotide 56gtggagttgt ggcggatctc 20 57 20 DNA Artificial Sequence AntisenseOligonucleotide 57 gtccttcatc cgcaggcagc 20 58 20 DNA ArtificialSequence Antisense Oligonucleotide 58 acagtccaca gacaagatct 20 59 20 DNAArtificial Sequence Antisense Oligonucleotide 59 gagctcccgc cgcagcttag20 60 20 DNA Artificial Sequence Antisense Oligonucleotide 60 ggagggattcgtcgagctcc 20 61 20 DNA Artificial Sequence Antisense Oligonucleotide 61atcttccact ggtaggactt 20 62 20 DNA Artificial Sequence AntisenseOligonucleotide 62 tgttgagcat cttccactgg 20 63 20 DNA ArtificialSequence Antisense Oligonucleotide 63 agctgctcca gcaaggagga 20 64 20 DNAArtificial Sequence Antisense Oligonucleotide 64 gctcgttcag ctgctccagc20 65 20 DNA Artificial Sequence Antisense Oligonucleotide 65 ttgccagccgggacacccag 20 66 20 DNA Artificial Sequence Antisense Oligonucleotide 66cgcagatagt actggtcttc 20 67 20 DNA Artificial Sequence AntisenseOligonucleotide 67 accgtggtga cccgcagata 20 68 20 DNA ArtificialSequence Antisense Oligonucleotide 68 cgagtcagaa gtgtgggaag 20 69 20 DNAArtificial Sequence Antisense Oligonucleotide 69 gtgatgggat cagagtcaaa20 70 20 DNA Artificial Sequence Antisense Oligonucleotide 70 ggagacttctacagggaccg 20 71 20 DNA Artificial Sequence Antisense Oligonucleotide 71gccacggtct ccataaattt 20 72 20 DNA Artificial Sequence AntisenseOligonucleotide 72 gcaaaagcaa catccacatc 20 73 20 DNA ArtificialSequence Antisense Oligonucleotide 73 tagagtgcag gatccagagc 20 74 20 DNAArtificial Sequence Antisense Oligonucleotide 74 attagttgca tgcaggagca20 75 20 DNA Artificial Sequence Antisense Oligonucleotide 75 agacagttttattgaattag 20 76 20 DNA Artificial Sequence Antisense Oligonucleotide 76cgagatagag ccactgtacg 20 77 20 DNA Artificial Sequence AntisenseOligonucleotide 77 tgccaccacc cccgggtgat 20 78 20 DNA ArtificialSequence Antisense Oligonucleotide 78 gttgttggtg gaacagtcca 20 79 20 DNAArtificial Sequence Antisense Oligonucleotide 79 tgcttaccgg tgctttttgc20 80 20 DNA Artificial Sequence Antisense Oligonucleotide 80 acatctcactcctcccggtg 20 81 20 DNA Artificial Sequence Antisense Oligonucleotide 81gaccctccaa gcgatcagct 20 82 20 DNA Artificial Sequence AntisenseOligonucleotide 82 aaaaagagga ccctccaagc 20 83 20 DNA ArtificialSequence Antisense Oligonucleotide 83 tgtgtcccct tttcacctgg 20 84 20 DNAArtificial Sequence Antisense Oligonucleotide 84 attaccaatg gagcatggca20 85 20 DNA Artificial Sequence Antisense Oligonucleotide 85 caacatggccaaaccccatg 20 86 20 DNA Artificial Sequence Antisense Oligonucleotide 86gcggcaggtc tccaggtctc 20 87 20 DNA Artificial Sequence AntisenseOligonucleotide 87 ttcccttcgg agagtagaga 20 88 20 DNA ArtificialSequence Antisense Oligonucleotide 88 tgcttgggaa atgcctgcaa 20 89 20 DNAArtificial Sequence Antisense Oligonucleotide 89 agctggatgc cagaaaggcc20 90 20 DNA Artificial Sequence Antisense Oligonucleotide 90 tggaagtagtggaagccagg 20

What is claimed is:
 1. A compound 8 to 50 nucleobases in length which istargeted to the 3′ UTR, an intron, an intron-exon junction, ornucleobases 106-1402 of the coding region of a nucleic acid moleculeencoding clusterin, wherein said compound specifically hybridizes withand inhibits the expression of clusterin.
 2. The compound of claim 1which is an antisense oligonucleotide.
 3. The compound of claim 2wherein the antisense oligonucleotide has a sequence comprising SEQ IDNO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 60, 61, 62, 63, 64, 65, 66, 67, 69, 70, 71, 72,73, 74, 76, 78, 79, 80, 82, 83, 84, 85, 86, 87, 88 or
 89. 4. Thecompound of claim 2 wherein the antisense oligonucleotide comprises atleast one modified internucleoside linkage.
 5. The compound of claim 4wherein the modified internucleoside linkage is a phosphorothioatelinkage.
 6. The compound of claim 2 wherein the antisenseoligonucleotide comprises at least one modified sugar moiety.
 7. Thecompound of claim 6 wherein the modified sugar moiety is a2′-O-methoxyethyl sugar moiety.
 8. The compound of claim 2 wherein theantisense oligonucleotide comprises at least one modified nucleobase. 9.The compound of claim 8 wherein the modified nucleobase is a5-methylcytosine.
 10. The compound of claim 2 wherein the antisenseoligonucleotide is a chimeric oligonucleotide.
 11. A compound 8 to 50nucleobases in length which specifically hybridizes with at least an8-nucleobase portion of an active site on a nucleic acid moleculeencoding clusterin.
 12. A composition comprising the compound of claim 1and a pharmaceutically acceptable carrier or diluent.
 13. Thecomposition of claim 12 further comprising a colloidal dispersionsystem.
 14. The composition of claim 12 wherein the compound is anantisense oligonucleotide.
 15. A method of inhibiting the expression ofclusterin in cells or tissues comprising contacting said cells ortissues with the compound of claim 1 so that expression of clusterin isinhibited.
 16. A method of treating an animal having a disease orcondition associated with clusterin comprising administering to saidanimal a therapeutically or prophylactically effective amount of thecompound of claim 1 so that expression of clusterin is inhibited. 17.The method of claim 16 wherein the disease or condition is ahypercholesterolemia.
 18. The method of claim 16 wherein the disease orcondition is a cardiovascular disorder.
 19. The method of claim 16wherein the disease or condition is a hyperproliferative disorder. 20.The method of claim 16 wherein the disease or condition is ahyperlipidemic disorder.